The world’s smallest vertebrate species of the genus Paedocypris: A new family of freshwater fishes and the sister group to the world’s most diverse clade of freshwater fishes (Teleostei: Cypriniformes)

Molecular Phylogenetics and Evolution 57 (2010) 152–175

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The world’s smallest vertebrate species of the genus Paedocypris: A new family of freshwater fishes and the sister group to the world’s most diverse clade of freshwater fishes (Teleostei: Cypriniformes) Richard L. Mayden a,*, Wei-Jen Chen b a b

Department of Biology, Saint Louis University, 3507 Laclede Ave., St. Louis, MO 63103-2010, USA Institute of Oceanography, National Taiwan University, No.1 Sec. 4 Roosevelt Rd., Taipei 10617, Taiwan

a r t i c l e

i n f o

Article history: Received 7 November 2009 Revised 29 March 2010 Accepted 2 April 2010 Available online 14 April 2010 Keywords: Paedocypris Smallest vertebrate Nuclear genes Phylogeny Cypriniformes

a b s t r a c t The genus Paedocypris has only recently been discovered and described and includes three species, all of which are miniature species and one, P. progenetica, is the smallest vertebrate species. Two previous studies investigating relationships of Paedocypris, based on either cytochrome b or morphology, placed the genus with Sundadanio and Danionella, two genera with miniature species in the formerly recognized family Cyprinidae. Our investigation of the phylogenetic relationships of Paedocypris using six nuclear genes and a broad survey of taxa in major lineages of the Cypriniformes identifies Paedocypris as a monophyletic group and the basal sister group to all Cypriniformes, not a species of the formerly recognized family Cyprinidae. These new relationships are also supported by previously proposed morphological characters but reinterpreted relative to ontogenetic hypotheses and outgroup comparisons used to determine synapomorphies. Miniaturization has occurred independently multiple times in the order, but mostly in the Rasborine Clade. Consequently, the hypothesis of a shared ancestral developmental truncation of multiple morphological features in genera with miniature species is rejected. While strong evidence exists for the new phylogenetic placement of Paedocypris as the sister group to the most diverse clade of freshwater fishes attempts to theorize more broadly as to evolutionary processes of miniaturization would be premature without more complete taxon sampling. Accompanying growing consistency of phylogenetic evidence of relationships in the Cypriniformes has come the consistent support of major clades within the previously recognized family Cyprinidae now recognized as a series of separate families, rendering the former Cyprinidae equivalent to Cyprinoidea. The revised family Cyprinidae includes species of the former subfamily Cyprininae, sister to Psilorhynchidae. The former subfamilies of Cyprininae, Acheilognathinae, Leuciscinae, and Gobioninae are elevated to families, and in keeping with consistency between phylogenetic relationships and classification the families Leptobarbidae and Tincidae are now recognized and the new families Tanichthyidae and Sundadanionidae are described. Paedocypris is recognized in a new superfamily, Paedocypridoidea, and family, Paedocyprididae. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction ‘‘Phylogenetic reconstruction is fundamental to comparative biology research . . . as the phylogeneticists’ conclusions (i.e., their phylogenetic inferences) become the comparative biologists’ assumptions. Consequently, the generation of robust phylogenetic hypotheses and the understanding of the factors influencing accuracy in phylogenetic reconstruction are crucial to evolutionary hypothesis testing.” Antonis Rokas and Sean B. Carroll (2005: 1337).

* Corresponding author. Fax: +1 314 977 3658. E-mail address: [email protected] (R.L. Mayden). 1055-7903/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2010.04.008

Evolutionary hypotheses and investigations are best conducted and tested within a phylogenetic framework depicting sister-group relationships of the targeted taxa (Wiley, 1981; Mayden, 1992). Conclusions regarding evolutionary mechanisms, developmental biology, adaptation, convergence, and/or parallel evolution demand a phylogeny of the species in question depicting sister-group relationships. In theory, the process of descent has produced a single evolutionary history of life or tree of species (Wiley, 1981), and one should be able to reconstruct this history using any number of heritable character types (e.g., DNA, proteins, morphology, etc.) and appropriate phylogenetic methodologies (Mayden and Wiley, 1992). With the coalescence of advancements in molecular biology, informatics, and computational biology there has emerged a new era of phylogenomics in systematic biology, wherein large

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numbers of nuclear and mitochondrial genomes or DNA sequences are now available for inferences of the tree of life (Chen et al., 2004; Chen and Mayden, 2010) and these trees can and should be used to examine a vast array of questions in comparative and evolutionary biology. Historically, limitations in developing strongly corroborated phylogenies of large clades centered on access to organisms and tissues (for morphological or molecular characters, respectively) and/or access to a diverse array of genes. With respect to molecular trees, until recently, the vast majority of phylogenetic inferences have derived from the maternally inherited mitochondrial genome. This latter constraint has been overcome recently for many groups of fishes, and other groups, with the development of several single copy nuclear genes appropriate for phylogenetic inference (Chen et al., 2004; Chen and Mayden, 2010), allowing researchers to expand beyond single gene phylogenies of the mitochondrion, or gene trees. Phylogenetic reconstructions of species serve as a critical framework with which scientists can better understand the evolution of biological attributes and use as excellent tools to facilitate the organization and interpretation of patterns and processing in education of the natural sciences. Likewise, phylogenies have become increasingly more important to the layperson via education to more clearly interpret various biological and geologically-linked (biogeography) aspects of the organisms. Optimization and reconstructions of qualities of taxa (species, genera, families, etc.) onto phylogenetic reconstructions and statistical tests of adaptation, origins of key features in radiations, rates of evolution, etc., however, are highly sensitive to several variables. Probably the one most important variable is taxon sampling, and in some cases as complete as possible (excluding extinct and unknown species) sampling of species that evolved from the hypothesized common ancestor to the group being examined (Frumhoff and Reeve, 1994; Hillis, 1996, 1998; Cunningham, 1999; Salisbury and Kim, 2001; Hillis et al., 2003; Felsenstein, 2004; Heath et al., 2008; but see Li et al., 2008). It is impossible to know before any study what taxa may possess in terms of particular attributes or where particular traits will finally resolve in a phylogeny, or how both of these will influence the results of optimization and statistical tests of relationships of such trait qualities at both ancestral and descendant nodes. Of course one may generate hypotheses of such things and test them following reconstructions and statistical optimizations. Thus, just as increased taxon sampling has been identified by empirical analyses to be a critical component in accurately reconstructing a tree, it necessarily follows that increased taxon sampling will provide more robust reconstructions of the evolution of traits, biogeographic patterns, and various other natural processes (Hillis, 1996, 1998; Heath et al., 2008; Mayden et al., 2008). In 2006 the world was reminded of the real significance of ‘‘descriptive science” and scientists involved with the inventories of biodiversity from so many areas around the planet that remain very poorly inventoried. From tannin-stained, highly acidic (ca. pH 3–5) peat swamp and flooded forests or blackwater swamps of southeast Asia, Kottelat et al. (2006) described the smallest vertebrate species living independently as an adult (Fig. 1A and B). One of the two described species, Paedocypris progenetica, matures at only 7.9 mm and is the smallest mature adult vertebrate species. The area and habitats where these miniature species were found had never before been inventoried so thoroughly and the radically different and new species discovered were placed in the newly described genus Paedocypris and allocated to the Order Cypriniformes and the formerly recognized family Cyprinidae (former family Cyprinidae recognized herein as superfamily Cyprinoidea, both groups hypothesized to be monophyletic. The superfamily Cyprinoidae also includes the model organism Danio rerio, Zebrafish, a species of the Rasborine Clade of the Cyprinoidea and one receiving

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enormous scientific attention for underlying genetic mechanisms controlling aspects of it’s growth and development, and for inferences in human biomedical research. The superfamily (and order) is also renowned for its great diversity of body size as well as many other biological attributes (Nelson, 2006; Froese and Pauly, 2009). Both extremes of the continuum of body size can be found in Cyprinoidae, with some reaching exceptional body sizes and prized for sport fishing, including many carp species (Cyprinus) and Masheers (Tor). The largest species include those of the genera Tor (275 cm, 54 kg) and Catla (182 cm, 38.6 kg) (Fig. 1C) (Froese and Pauly, 2009). With the discovery and description of Paedocypris progenetica (Kottelat et al., 2006) and P. micromegethes, adult body size in the superfamily (and order) ranges from 7.9 to 2750 mm, or about 2.5 orders of magnitude. The habitats wherein these authors collected these enigmatic fishes have been grossly under explored, as have been many other areas around the planet, but continue to reveal many new species, evolutionary novelties of morphologies, physiologies, and genetic information, some of which has the potential to be critical importance in resolving fundamental evolutionary, and possibly other questions (e.g., Britz et al., 2009). At the same time that these and other unique and poorly inventoried forests are rapidly disappearing due to human activities, they hold elements of life never before seen or described and life forms or interactions as fundamental as the discovery of the medicinal properties of the rosy periwinkle, Catharanthus roseus, and Thermus aquaticus, the bacterium that permitted thermal cycling and revolutionized DNA applification. Continued inventory efforts have identified additional miniature species of this genus that remain to be described from other areas of southeast Asia (Britz, pers. commun.). In the descriptions of the new species of Paedocypris the authors chose not to comment on the evolutionary relationships of the species using the morphological characters available to them at the time. Subsequent molecular and morphological investigations by Rüber et al. (2007) and Britz and Conway (2009), respectively, examined the relationships of Paedocypris. In both instances these miniature fish species were determined to be in a general group of Cyprinidae known as the ‘‘Rasborines” a clade that has never been demonstrated to be monophyletic. Rüber et al. (2007) turned to a molecular reconstruction of relationships with the Cyprinidae that would be independent of miniaturization and analyzed complete sequences of cytochrome b, the most commonly used mitochondrial gene in molecular systematics of fishes and readily available for many species, as a means to investigate both the evolution of miniaturization and date the origin of these species. Their study identified Paedocypris as a monophyletic group sister to another group of miniature rasborine cyprinids from southeast Asia in the genus Sundadanio. While the evolution of miniaturization was hypothesized by Rüber et al. (2007) to have occurred in the common ancestor to these two genera and this clade was found to be the basal sister group in the family Cyprinidae, they also identified multiple instances in the family where a truncated development, leading to miniaturization in adults, had occurred in their evolution, and hence, multiple cases of parallel evolution of this developmental phenotype. A minimum age of divergence for Paedocypris and Sundadanio was estimated at ca. 24 MYBP. Britz and Conway (2009) presented a detailed description of much of the macroanatomy of Paedocypris, discussed the hypothesized developmental truncations for these species and other miniatures, and hypothesized relationships of the genus within the order and family using only morphological characters. Their phylogenetic analysis was restricted to anatomical characters. The authors recognized problems with Paedocypris not possessing some appropriate morphological synapomorphic characters for the order Cypriniformes and the family Cyprinidae, and justified this mismatch of synapomorphies as a result of the evolution of miniaturization,

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Fig. 1. Extremes in body size of adults of species of the Order Cypriniformes. (A and B) Adult specimen of Paedocypris (69 mm SL) (photographs by Dr. Kobayashi and used with permission). (C) Adult specimen of Catlocarpio siamensis, one of the largest species of Cypriniformes (172 cm, 102 kg) (photograph by Dr. Zeb Hogan and used with permission).

truncated development, and modifications to anatomy, occurrences that apparently did not happen in its purported sister group Sundadanio (or Danionella). The phylogenetic analysis was for all intents and purposes limited to a hypothesis of relationships not among a random or near random set of Cypriniformes taxa but was from among a select group of taxa that were identified a priori as miniaturized species sensu criteria of Weitzman and Vari (1988) (species reproducing 620 mm, or if reproductive information are not available, species less than 26 mm). Justification for the limited taxon sampling rested on the assumption that miniature cyprinids descended from a common ancestor and within the ‘‘clade” there had been a pattern of progressively developed smaller body sizes. Hence, Paedocypris would have to be closely related to other miniature species of Cyprinidae. In their study they argued for a sistergroup relationship between Paedocypris and Danionella and this clade sister to Sundadanio. Britz and Conway (2009) acknowledged their difficulty in placing Paedocypris in a phylogenetic system due to issues associated with developmental truncation, invoked assumptions of relatedness amongst known miniature cyprinids, and ultimately discovered difficulties in identifying sister-group relationship of the genus within Cypriniformes and Cyprinidae because of long known problematic diagnostic features for the various subfamilies and genera. Thus, two recent phylogenetic evaluations of Paedocypris, one using a mitochondrial gene and

the other a suite of anatomical features, have converged on nearly identical sister-group relationships for the smallest vertebrate species with another miniaturized cyprinid genus. These hypotheses for miniaturization occurring independently in other lineages within the rasborine cyprinids represents a convergence of hypotheses optimal for further study as to the developmental and genetic origins involved in miniaturization. Recent phylogenetic studies of Cypriniformes fishes from The Cypriniformes Tree of Life initiative (CToL; www.cypriniformes.org) have revealed five critical findings relevant to the current molecular hypothesis as to the origin of the smallest vertebrate species (Rüber et al., 2007). Phylogenetic inferences as to the relationship of this and other species are highly sensitive to (1) saturation of third base positions in protein coding genes (e.g., cytochrome b) (hence, possible long branch attraction; see Fig. 1 in Saitoh et al., 2006), (2) reliance upon single genes, (3) taxon sampling, (4) real influences on topologies based on statistically significant GC content bias of genes, and (5) the importance of reliance upon diverse genetic markers (Mayden et al., 2007, 2008, 2009; Chen et al., 2008; Chen and Mayden, 2009). With the growing development of many new molecular markers from the nuclear genome (Chen et al., 2008) and use of traditional markers from the mitochondrial genome (Mayden et al., 2007, 2008) the era of phylogenomics (Chen and Mayden, 2010) offers considerably greater

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opportunities in searching tree space for more accurate estimates of a phylogenetic history necessary for evolutionary inferences, such as in this case body size evolution and possible developmental truncation events. Herein, we present new, and well corroborated, findings as to the phylogenetic relationship of the world’s smallest vertebrate that are significantly different from and reject previous hypotheses. Evidence derived from six nuclear genes supports the hypothesis that species of Paedocypris are not cyprinoids. The nuclear genome also provides further evidence for multiple and widely divergent, independent evolutionary origins of miniaturization in cypriniform fishes. This evolutionary phenomenon may have arisen in different manners as previously discussed by McClain and Boyer (2009) for some vertebrates, but its origin and maintenance is hypothesized to be a lifestyle of lineages living in or associated with highly acidic freshwater habitats. We hypothesize that the relationships resolved by Rüber et al. (2007) are the result of relying on a single, relatively rapidly evolving protein-coding mitochondrial gene. The difficulties encountered by Britz and Conway (2009) in their search for the phylogenetic placement of Paedocypris derives from a reliance upon the idea of progressive evolution in miniaturization via a ‘‘step by step” process and idealistic morphology, only exacerbated by their limited selection of species assumed to be related to Paedocypris, the conflation of miniaturization as a causative explanation for the developmental admixture of synapomorphic and pleisomorphic traits not characteristic of either Cypriniformes or the formerly recognized family Cyprinidae and the lack of some of these characteristics because the species is neither a cyprinid nor a cyprinoid. We argue herein that a more parsimonious explanation for the observed admixture of synapomorphic anatomical characters in Paedocypris is that the species of Paedocypris are not descendants of the common ancestor to the current conception of Cypriniformes. Unlike previous reports, the nuclear genome does not support Paedocypris as closely related to other miniature ‘‘rasborin-like” fishes (Sundadanio or Danionella) within Cyprinoidea. Rather, the genus forms the basal sister group to the entire order, inclusive of not only the great diversity in the formerly recognized family Cyprinidae but also the other families Catostomidae, Gyrinocheilidae, Cobitidae, Ellopostomatidae, Ballitoridae, and Vaillantellidae. We present evidence that this sister-group relationship to the clade Cyprinoidea plus Cobitoidea is also supported by a reevaluation of the anatomical characters presented by Britz and Conway (2009). All published hypotheses as to the relationships of Paedocypris are rejected; however, unpublished data based on whole mitochondrial genomes (Miya et al., 2007) does place Paedocypris as the sister group to Cyprinoidea, a hypothesis we cannot reject with our data but one that is less probable. Regardless of which of these enormous and powerful data sets is evaluated, they both clearly reject the idea assumed by progressive evolution of singular evolution of miniaturization in the Cyprinoidea. The morphological evidence bearing on this question unequivocally supports Paedocypris as the sister group to all Cypriniformes and rejects the hypothesis of Paedocypris as the sister group to the Cyprinoidea. Our new phylogenetic reconstruction requires a renewed evaluation of the evolution of these diverse and important fishes, the evolution of miniaturization, and the establishment of a new classification for Paedocypris. Herein, we also reemphasize the non-monophyly of the formerly recognized Cyprinidae relative to Psilorhynchidae and note that many of the usual subfamilies formerly in Cyprinidae and monophyletic groups formerly recognized within Cyprinidae should be considered families; the family Cyprinidae should hereafter be restricted to the former subfamily Cyprininae. Without these changes and the description of new families (Sundadanidae, Tanichthidae, Paedocyprididae), elevation of formerly defined and available names within the superfamily to the family level, and a new classification for Paedocypris the placement

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of this genus in Cyprinidae renders the superfamily Cyprinoidea and the family ‘‘Cyprinidae” polyphyletic. 2. Materials and methods 2.1. Taxa and classification Classification of major lineages within the Cypriniformes follows recent changes at the family level by Chen and Mayden (2009) and Chen et al. (2009). Our use of the term ‘‘miniature” for fishes follows the classification by Weitzman and Vari (1988) and refers to an adult body size of 26 mm standard length (SL) or less (also used by Kottelat et al., 2006; Rüber et al., 2007; Britz and Conway, 2009). Our sampling of taxa included all major lineages within the Cypriniformes, including members from the various newly erected families within the Cyprinoidea (Chen and Mayden, 2009) and Cobitoidea (Chen et al., 2009); however, we provided more samples of species from the ‘‘Rasborine Clade” (species in the formerly recognized family Cyprinidae, often referred to as ‘‘rasborins”) because it is in this general ‘‘clade” that most miniature cypriniform fishes have been recognized. Furthermore, in the previous phylogenetic analyses involving Paedocypris by Rüber et al. (2007), and in descriptive and comparative studies of Paedocypris (Britz and Conway, 2009), the taxon has been either compared with/to species traditionally referred to ‘‘rasborins” or found to be closely related to some species of the clade (e.g., Sundadanio) also included in herein. 2.2. Samples, genes, and sequences A total of 85 samples were included for investigation, representing both the Cobitoidea and Cyprinoidea and all of the recognized families and subfamilies in the order Cypriniformes. The analytical dataset was composed of DNA sequences of six targeted nuclear gene loci from these taxa, and four outgroup taxa (Gonorynchus greyi, Pseudobagrus tokiensis, Phenacogrammus interruptus, Chalceus macrolepidotus). Several sequences used in this study were previously described in Chen et al. (2008, 2009), Chen and Mayden (2009), and Mayden et al. (2009). New sequences for all six genes were obtained for nine samples of eight taxa (Boraras merah, Chela dadiburjori, Danio erythromicron, Danio margaritatus, Microrasbora nana, Microrasbora rubescens, Paedocypris sp. (2 specimens), Sundadanio axelrodi) and are deposited in GenBank. GenBank accession numbers of sequences used in this study appear in the Appendix A. Protocols for collecting new DNA data follow those outlined in Chen et al. (2008). 2.3. Phylogenetic inferences Phylogenetic analyses were conducted using the partitioned Maximum Likelihood (ML) method and partitioned Bayesian approach (BA) as implemented in the parallel version of RAxML 7.0.4 (Stamatakis, 2006) and MrBayes 3.1.1 (Huelsenbeck and Ronquist, 2001), respectively. All analyses included assignment of 18 partitions with respect to the gene and the codon positions (first, second, and third position of protein-coding genes, respectively). Search for optimal ML trees and Bayesian analyses were performed by a high performance cluster computing facility (20 nodes) at Saint Louis University. Nodal support was assessed with bootstrapping (BS) (Felsenstein, 1985) with the Maximum Likelihood (ML) criterion, based on 1000 pseudo-replicates and the resulting a posteriori probabilities from partitioned BA. The MLBS analyses (through analyses using RAxML web-servers) (Stamatakis et al., 2008) were conducted with the CIPRES cluster (CIPRES Portal 1.13, http://www.phylo.org/sub_sections/portal/) at the San Diego

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Table 1 Support for alternative hypotheses depicting the sister-group relationship of Paedocypris assessed by the procedures followed by Maximum likelihood (ML) bootstraps (BS), posteriori probability using Bayesian approach (PP), and Shimodaira–Hasegawa’s test (SH) under optimal criterion of tree reconstruction of ML. Hypothesisa

I II III IV V

Matrix 1 (equal weighting)

Matrix 2 (partial RY-coding)

BS (%)

PP

SH (p-value)

BS (%)

PP

SH (p-value)

99 1 0 0 0

1.00 0 0 0 0

Best 0.3205 0.0003 <0.0001 <0.0001

31 69 0 0 0

0.87 0.13 0 0 0

Best 0.7471 0.0149 <0.0001 <0.0001

a Five alternative hypotheses are: (I) Paedocypris sister group to all cypriniforms (Fig. 2, this study); (II) Paedocypris sister group to all cyprinoids (based on whole mitochondrial genome data from Miya et al. unpubl; Miya et al., 2007); (III) Paedocypris sister group to Sundadanio (based on mitochondrial cytochrome b sequence from Rüber et al., 2007); (IV) Paedocypris sister group to Danionella (based on morphological data from Britz and Conway, 2009); (V) Sundadanio sister group to Paedocypris plus Danionella (based on morphological data from Britz and Conway, 2009).

Supercomputer Center. A test of homogeneity of base frequencies across taxa was conducted for each gene and codon position separately using the v2 test implemented in PAUP*. As suggested by Chen and Mayden (2009) if tests of base composition stationarity reveal bias across taxa at variable sites or sites at a particular codon position these sites should be examined using RY-coding schemes in addition to other analyses. We constructed an additional dataset (matrix 2) in which the nucleotides A and G and the nucleotides T and C at the third codon position of these four genes were converted into purine (R) and pyrimidine (Y), respectively. Analyses based on this matrix are herein referred to as partial RY-coding analysis. Detailed protocols for phylogeny reconstruction follow those outlined in Chen and Mayden (2009) and Chen et al. (2009). In addition, alternative published hypotheses depicting the sister-group relationship of Paedocypris (Table 1) were tested using the approach proposed by Shimodaira and Hasegawa (1999) (SH) for ML analysis as implemented in PAUP*. Four constrained analyses corresponding to those previous hypotheses (Table 1) were conducted. The tree scores ( ln likelihood) resulting from these constrained analyses were then compared to the tree score of the best trees (Fig. 2) under ML criterion. Differences in scores between tree topologies (best and constrained) were statistically evaluated via a resampling approach (RELL) with 10,000 bootstrap replications. 2.4. Morphological characters Morphological characters related to the placement of Paedocypris relative to other Cypriniformes were derived from the descriptions and discussions by Britz and Conway (2009) as to the phylogenetic placement of the genus, including those characters listed by the former authors that were derived from Fink and Fink (1981) and Siebert (1987). These characters were optimised on a phylogenetic tree of many more cypriniform taxa consistently supported in studies by Mayden et al. (2007, 2008, 2009), Chen and Mayden (2009), Chen et al. (2008), and Chen et al. (2009) and illustrated herein (Fig. 3) as a reduced cladogram to demonstrate the logical consistency of morphological character evolution with the new placement of Paedocypris. 3. Results 3.1. Genes, sequence variation, and base composition A total of 5733 bp were aligned for the exon regions of the six nuclear genes for 85 taxa (including 4 outgroups) sampled in this study. The length of aligned sequences from each locus was 1497 bp (RAG1), 819 bp (RH), 849 bp (IRBP), 846 bp (EGR1), 816 (EGR2B), and 906 (EGR3). Few indels were needed in adjusting sequence alignment of the EGR genes, but the alignment was unam-

biguously achieved followed by triplet codes for amino acids. Of the 5733 nucleotides, 2632 were variable and 2562 of these were phylogenetically informative. As in Chen et al. (2008), no clear saturation plateau on substitutions in transitions at the third codon position of the six nuclear genes used here appeared in sequences from all the major cypriniform lineages (see Fig. 2 in Chen et al., 2008). However, tests of base composition stationarity revealed that variable sites and sites at the third codon position in RAG1, rhodopsin, EGR2B, and EGR3 sequences exhibit significant base composition bias across taxa. 3.2. Phylogenetic relationships based on molecular characters Maximum likelihood analyses of the six nuclear genes (RAG1, rhodopsin, IRBP, EGR1, 2B and 3) implementing equal weighting and partial RY-coding procedures, consisting of 5733 aligned nucleotides, yielded identical and strongly supported topologies for members of the order (Fig. 2). Nodal support can vary depending on the analysis. The order is strongly supported as monophyletic with Paedocypris forming a monophyletic group sister to all remaining members of the Cypriniformes, a result that received excellent bootstrap support (Fig. 2). The two traditionally recognized superfamilies, Cyprinoidea and Cobitoidea, were resolved as monophyletic groups with strong support. Within the Cobitoidea all of the families were monophyletic and Catostomidae was resolved as sister to the Gyrinocheilidae plus a monophyletic group of five families currently referred to as loaches (Fig. 2). Within the Cyprinoidea, the monophyletic families Cyprinidae plus Psilorhynchidae formed a clade sister to remaining families. The ‘‘Rasborine Clade,” a group traditionally containing all of the miniature cypriniform species and formerly hypothesized to contain Paedocypris, formed the basal sister group to a clade including Leptobarbidae plus Sundadanionidae (another clade with miniature species), as well as remaining families. The family Leuciscidae formed the sister group to the family Gobionidae and this clade was sister to Tanichthyidae. The ‘‘Cultrine Clade” formed the sister group to Acheilognathidae plus Tincidae plus Tanichthyidae, Gobionidae, and Leuciscidae. Tests of alternative phylogenetic relationships of Paedocypris (Table 1) revealed that the phylogenetic resolution depicted herein (Fig. 2) was the best hypothesis. All previous hypotheses of relationships of Paedocypris were rejected except wherein Paedocypris forms the sister group to all Cyprinoidea (see Miya et al., 2007; Fig. 2). Hypotheses of Paedocypris as either sister to Sundadanio (Rüber et al., 2007), Danionella (Britz and Conway, 2009), and Sundadanio sister to Paedocypris plus Danionella (Britz and Conway, 2009) were all significantly incongruent with genetic variation and phylogenetic interpretation of the results presented herein (Fig. 2). We reject the sister-group relationship of Paedocypris with Cyprinoidea on the basis of the highly congruent synapomorphic

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Fig. 2. Phylogenetic tree depicting relationships among taxa from the Cypriniformes. Relationships inferred using partitioned ML analysis of 5733 aligned nucleotides from six nuclear gene loci based on data matrix 1 (all of nucleotide sequences or equal weighting data, A–left topology) and matrix 2 (partial RY-coding data, B–right topology). ML score of the tree is 95,608.114731 and 63,979.766547 for analysis 1 and 2, respectively. Branch lengths of left topology are proportional to inferred character substitutions under GTR + G + I model. Numbers on branches are ML bootstrap values; those below 50% are not shown. Bold branches on topologies indicate robust nodes with a posteriori probabilities from partitioned Bayesian analysis P0.95. The targeted taxa in this study, Paedocypris, Sundadanio and Danionella are marked in bold. Asterisk () indicates taxa with miniature adult body size (26 mm SL or below; sensu Weitzman and Vari, 1988). Classification follows Šlechtová et al. (2007) and Chen and Mayden (2009). The elevated families Acheilognathidae, Leuciscidae, Gobionidae Leptobarbidae (Leptobarbus) from Leptobarbi recognized by Bleeker (1864:116 in second part of Bleeker (1863b–1864) (Type species Leptobarbus) (all former subfamilies of Cyprinidae) and new families, Paedocyprididae (Paedocypris), Tanichthyidae (Tanichthys) and Sundadanionidae (Sundadanio) are herein recognized based on phylogenetic relationships of these natural groups.

Condition in Paedocyprisa

Interpretation of morphological characters herein with character number and state in parentheses. Characters and states are provided on the phylogeny (Fig. 3) and illustrated in Fink and Fink (1981), Britz and Conway (2009), and Fig. 4, herein

Proposed Synapomorphy for Otophysi 1 Weberian apparatus

Present

Synapomorphy for Otophysi

Proposed Synapomorphies for Cypriniformes 2 Bony kinethmoid element [1]

Present as cartilage only

No.

Taxon and hypothesized synapomorphic character or complex

Present Present

Synapomorphy for Cypriniformes (4A)

Present

Synapomorphy for Cypriniformes (5A)

Absent

7

Palatine process that abuts against the mesethmoid [21]

Only small ‘‘nubbin” of membrane bone projecting dorsally towards mesethmoid

8

Palatine/endopterygoid articulatory facet [22]

Absent

9

Ectopterygoid not overlapping the palatine anteriorly [25] Premaxilla extending farthest dorsally adjacent to the midline [37]

Ectopterygoid absent

Synapomorphy for Cobitoidea + Cyprinoidea (6A). Preethmoid evolves in common ancestor to this clade. The preethmoid (Fig. 4) may be either cartilage or ossified Small palatine process is precursor to the enlarged process (Fig. 4D and E – PP and PAL, herein) that is synapomorphic for Cobitoidea + Cyprinoidea (7B). Thus the small nubbin is interpreted as synapomorphic for Paedocypris (Cobitoidea + Cyprinoidea) (7A) and is retained in this condition in Paedocyprididae and Paedocypridoidea. Synapomorphy for Cypriniformes. Further enlargement of the process (7B) is synapomorphic for Cyprinoidea + Cobitoidae Lack of palatine/endopterygoid articulatory facet is hypothesized as plesiomorphic condition and is retained in Paedocypris, Paedocyprididae, Paedocypridoidea. The articulatory facet (Fig. 4D and E – ENP and PAL, herein) is a synapomorphy for Cobitoidea + Cyprinoidea (8A) Autapomorphy for Paedocypris, Paedocyprididae, Paedocypridoidea (9A)

3 4 5 6

10

11

Jaw teeth absent* [42]

12 13

Teeth on second and third pharyngobranchials and basihyal absent* [47] Two posterior pharyngobranchial toothplates absent [48]

14

Toothplate associated with basibranchials 1–3 absent* [50]

15

Epurals two or fewer* [115]

16

*

Adipose fin absent [125] *

17

Number of postcleithra reduced to one [96];

18

Maxillary barbels

19

Masticatory plate on basioccipital present sensu, Howes, 1981

Absent. Premaxilla of Paedocypris is similar in length to the maxilla but exhibits only a small expansion of its medial-most tip and only a short ascending process on the premaxilla is bound to the kinethmoid cartilage True, homoplasious sensu Fink and Fink (1981) but synapomorphy for Cypriniformes True, homoplasious sensu Fink and Fink (1981) but synapomorphic for Cypriniformes True, homoplasious sensu Fink and Fink (1981) but synapomorphic for Cypriniformes True, homoplasious sensu Fink and Fink (1981) but synapomorphic for Cypriniformes True, homoplasious sensu Fink and Fink (1981) but synapomorphic for Cypriniformes True, homoplasious sensu Fink and Fink (1981) but synapomorphic for Cypriniformes Postcleithra absent in Paedocypris (sensu Britz and Conway, 2009) and proposed by these authors as a more derived state for lineage Maxillary barbels hypothesized to be lost in Paedocypris and apomorphic for genus (sensu Britz and Conway, 2009). Apomorphic for Cypriniformes sensu Fink and Fink (1981). True

Small and short premaxillary process at midline is precursor to enlarged process that is synapomorphic for Cobitoidea + Cyprinoidea (10B). Thus, the smaller expansion is interpreted as synapomorphic for Paedocypris (Cobitoidea + Cyprinoidea) and is retained in this condition in Paedocyprididae and Paedocypridoidea (10A). Synapomorphy for Cypriniformes Synapomorphy for Cypriniformes (11A) Synapomorphy for Cypriniformes (12A) Synapomorphy for Cypriniformes (13A) Synapomorphy for Cypriniformes (14A) Synapomorphy for Cypriniformes (15A) Synapomorphy for Cypriniformes (16A) Autapomorphy for Paedocypris, Paedocyprididae, Paedocypridoidea (17B)

Lack of maxillary barbels is hypothesized as the plesiomorphic ancestral condition for Cypriniformes (inclusive of Paedocypris as sister to remaining Cypriniformes) and evolved independently in ancestor to Cyprinoidea (18A*) and ancestor within in Cobitoidea above Catostomidae and Gyrinocheilidae (18A*). Thus, absensce of maxillary barbels is hypothesized as a plesiomorphic condition in Paedocypris Masticatory plate on basioccipital (Fig. 4H – PPD, herein) is hypothesized to be synapomorphic for Cypriniformes (19A) with further modification of this structure in Catostomidae as the

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Fifth ceratobranchial enlarged extending much further dorsally than other ceratobranchials [52] Teeth on the fifth ceratobranchial ankylosed to the bone [53] Lateral process of second centrum elongate and extending well into body musculature [84] Preethmoid [4]

Cartilage is a precursor to ossified kinethmoid that is synapomorphic for Cobitoidea + Cyprinoidea (2B). Thus, the cartilage kinethmoid is interpreted as synapomorphic for Paedocypris (Cobitoidea + Cyprinoidea) (2A) and is retained as cartilage in Paedocyprididae and Paedocypridoidea Synapomorphy for Cypriniformes (3A)

158

Table 2 Synapomorphic and apomorphic (some proposed autapomorphic homoplasious) characters for Ostariophysi, Cypriniformes, and within the Cyprinoidea and Cobitoidea from Fink and Fink (1981), their conditions in Paedocypris, as identified by Britz and Conway (2009), and our interpretation of the character transformation or optimization based on the new molecular phylogenetic hypothesis for Paedocypris.

20

Posterior extension of basiocciptial forming the pharyngeal process of basioccipital (sensu Winterbottom, 1974, Fink and Fink, 1981) forming synapomorphy of Cypriniformes (Mayden, Pers. Obs.)

Proposed Synapomorphies for Cobitoidea plus Cyprinoidea Proposed Synapomorphies for Cyprinoidea (see Character 18 also) 21 Absence of an uncinate process on EB1 and EB2§ Proposed synapomorphy for Cyprinidae sensu Fink and Fink (1981) (= Cyprinoidea sensu Chen and Mayden, 2009) 22 Absence of PB1§

Absent

See Characters 6, 8, and 20 above Presence of uncinate process on EB1 and EB2 synapomorphic for Cyprinoidea (21A)

PB1 absent in Paedocypris

Absence of PB1apomorphic for Cyprinoidea (22A*). Independently lost in Paedocypris, Paedocyprididae, Paedocypridoidea (22A*) The interorbital septum being formed by the orbitosphenoid and parasphenoid is more widespread in Cypriniformes than hypothesized (Cavender and Coburn, 1992), being found in many species of both Cobitoidea and Cyprinoidea, and is hypothesized herein to be a synapomorphy for this combined clade (23A). However, the septum can be restricted to only the orbitosphenoid in some Cyprinoidea (Fig. 4B, F, and I – ORS, herein). The lack of the septum in Paedocypris is hypothesized herein to be plesiomorphic. However, in Britz and Conway (2009) a partial interorbital septum is formed by a ventral projection of the orbitosphenoid, a condition that could be interpreted as a precursor to the full development of the septum by both the orbitosphenoid and parasphenoid Autapomorphy for Paedocypris, Paedocyprididae, Paedocypridoidea (24A) Autapomorphy for Paedocypris, Paedocyprididae, Paedocypridoidea (25A) Deep, well-developed subtemporal fossa (Fig. 4A and J – PTF, herein) hypothesized herein to be synapomorphic for Cyprinoidea (26A), a condition lacking and interpreted as plesiomorphic in Paedocypris The anterior opening to the trigemino-facial foramen in cyprinoids is usually bound by the prootic (posterior rim) and pterosphenoid (anterior rim) (Fig. 4I, K and L – FVII, herein) but can be contained completely within the prootic (Fig. 4, herein). The absence of the foramen in Paedocypris is herein interpreted as a plesiomorphy and the presence of the foramen as described above is a synapomorphy for Cyprinoidea (27A) The fused second and third vertebrae is hypothesized to be the synapomorphic condition for Cyprinoidea (28A) and the condition in Paedocypris is plesiomorphic

Interorbital septum formed both by the orbitosphenoid and parasphenoid

Interorbital septum absent in Paedocypris

24 25 26

Loss of contact between infraorbital 5 and supraorbital Presence of an opercular canal Deep, well-developed subtemporal fossae

27

Anterior opening of the trigemino-facial chamber positioned between the prootic and the pterosphenoid

Infraorbital 5 absent in Paedocypris Sensory canals absent in Paedocypris Absent, hypothesized by Britz and Conway (2009) as absent due to developmental truncation Anterior opening determined to be absent by Britz and Conway (2009) due to developmental truncation

28

Fused second and third Weberian vertebrae

29

Overlap of PB2 by PB3

30

‘‘PB2 and 3 at the same level and confluent with each other and EB4” Britz and Conway (2009)

Absent, hypothesized by Britz and Conway (2009) as absent due to developmental truncation Absent, hypothesized by Britz and Conway (2009) as absent due to developmental truncation Present

Overlap of PB2 by PB3 synapomorphy for Cyprinoidea (29A)

Autapomorphy for Paedocypris, Paedocyprididae, Paedocypridoidea (28A)

True True

Autapomorphy for Paedocypris, Paedocyprididae, Paedocypridoidea (31A) Autapomorphy for Paedocypris, Paedocyprididae, Paedocypridoidea (32A)

True

Autapomorphy for Paedocypris, Paedocyprididae, Paedocypridoidea (33A)

True

Autapomorphy for Paedocypris, Paedocyprididae, Paedocypridoidea (34A)

True

Autapomorphy for Paedocypris, Paedocyprididae, Paedocypridoidea (35A)

159

(continued on next page)

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Uncinate processes lacking on EB1 and EB2 in Paedocypris

23

Synapomorphies of Paedocyprididae (sensu Britz and Conway, 2009 for Paedocypris) 31 Postcleithra are absent** [96] 32 Three uppermost pectoral-fin rays hypertrophied in males and articulating with likewise hypertrophied dorsal-most pectoral radial, which is very tightly associated with the second pectoral radial   33 Basipterygia of pelvic girdle in males hypertrophied with a large flange of membrane bone directed anterodorsally toward the outer arm of the os suspensorium   34 Dorsal and ventral hemitrichs of first pelvic-fin ray in males hypertrophied with additional anterior flanges that support keratinized skin pads   35 Presence of hemal spines in the abdominal region starting with vertebra 7  

palatal organ in all species (19B) and horny plate in some (Eastman, 1977); this plate is reduced in size in the common ancestor to Cobitoidea (19C), and lost above the Gyrinocheilidae plus other Cobitoidea (19D). Synapomorphy for Cypriniformes Pharyngeal process of basioccipital (Fig. 4G, H, and J – PPBO, herein) interpreted as synapomorphic for Cyprinoidea + Cobitoidea (30A) (Mayden, Pers. Obs.); absence in Paedocypris is hypothesized to be the retention of the plesiomorphic condition of no posterior process on the basioccipital

160

Table 2 (continued) No.

Taxon and hypothesized synapomorphic character or complex

Proposed synapomorphies for Paedocypris plus Danionella 36 EB5 articulates not only with the levator process of EB 4 but also with the tip of CB5 (Character 9 Britz and Conway, 2009)   37 PB 3 and EB4 connected via a continuous cartilage (Character 10 Britz and Conway, 2009)   38 Neural spine on fourth neural arch (Character 11 Britz and Conway, 2009)   39 Parietal absent (Character 12 Britz and Conway, 2009)   40

Interpretation of morphological characters herein with character number and state in parentheses. Characters and states are provided on the phylogeny (Fig. 3) and illustrated in Fink and Fink (1981), Britz and Conway (2009), and Fig. 4, herein

True

Independently derived in miniature ancestral species, one for Paedocypris (36B*) and an ancestral species of Danionella (36B*) Independently derived in miniature ancestral species, one for Paedocypris (37B) and an ancestral species of Danionella (37B*) Independently derived in miniature species, one in ancestor for Paedocypris (38B*) and an ancestral species of Danionella (38B*); presence is 38A Independently lost in miniature species, one in ancestor for Paedocypris (39B*) and an ancestral species of Danionella (39B*) Independently lost in miniature species, one in ancestor for Paedocypris (40B*) and an ancestral species of Danionella (40B*) Independently lost in miniature species, one in ancestor for Paedocypris (41B*) and an ancestral species of Danionella (41B*)

True True True True True Absent from their paper

Proposed synapomorphies of Paedocypris, Danionella, and Sundadanio 42 Concha scaphii reaches all the way to the end of the processus ascendens of the scaphium or in other words, there is no recognizable processus ascendens (Character 5 Britz and Conway, 2009)   43 Inner arms of ossa suspensoria are fused in their proximal portion, greatly elongated, following the curvature of the anterior chamber of the swimbladder and extending posteriorly to about half of its length (Character 6 Britz and Conway, 2009)   44 Gap between enlarged neural arches 3 and 4 filled by extensive development of cartilage (Character 7 Britz and Conway, 2009)   45 Tripus hypertrophied and unusually elongate (Character 8 Britz and Conway, 2009)   Bones lost only in Paedocypris, Danionella, and Sundadanio (from Britz and Conway, 2009) 46 Vomer (Character 1 Britz and Conway, 2009)

True

47

Ectopterygoid (Character 2 Britz and Conway, 2009)

True

48

Post-cleithrum (Character 3 Britz and Conway, 2009)

True

49

Post-temporal (Character 4 Britz and Conway, 2009)

True

Features found only in Paedocypis (from Britz and Conway, 2009) 50 Intercalarium reduced to the manubrium only and existing as a small splint of a bone in the interossicular ligament and far removed from its associated centrum 51 Hypertrophied first pectoral radial and three uppermost pectoral fin radials 52 Hypertrophied anterodorsally directed basipterygium 53 Hypertrophied first pectoral fin ray 54 Large dorsally directed triangular process on the lateral face of the outer arm of the os suspensorium 55 Haemal spines in abdominal region 56 Soft tissue structures as a prepelvic keratinized pad 57 Keratinized flanges of skin covering the enlarged first pelvic ray 58 Enlarged genital opening forming a bag around the first anal-fin rays (Kottelat et al., 2006)

True

Independently derived in miniature ancestral species, one for Paedocyprididae (42B*) and species ancestral to Sundadanio and Danionella (42B*)

True

Independently derived in miniature ancestral species, one for Paedocyprididae (43B*) and species ancestral to Sundadanio and Danionella (43B*)

True

Independently derived in miniature species ancestral to Sundadanio and Independently derived in miniature species ancestral to Sundadanio and

True

ancestral species, one for Paedocyprididae (44B*) and Danionella (44B*) ancestral species, one for Paedocyprididae (45B*) and Danionella (45B*)

Independently derived in miniature ancestral species, one for Paedocyprididae (46B*) and species ancestral to Danionella and Sundadanio (46B*) Independently derived in miniature ancestral species, one for Paedocyprididae (47B*) and species ancestral to Danionella and Sundadanio (47B*) Discussed above for Character 31. Independently derived in miniature ancestral species, one for Paedocyprididae (48B*) and species ancestral to Danionella and Sundadanio (48B*) Independently derived in miniature ancestral species, one for Paedocyprididae (49B*) and species ancestral to Danionella and Sundadanio (49B*)

True

Apomorphy for Paedocypris

True

Apomorphy for Paedocypris

True True True

Apomorphy for Paedocypris Apomorphy for Paedocypris Apomorphy for Paedocypris

True True True True

Apomorphy Apomorphy Apomorphy Apomorphy

for for for for

Paedocypris Paedocypris Paedocypris Paedocypris

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41

Hypobranchial 3 absent (Character 13 Britz and Conway, 2009)   Supraneurals posterior to superneural 3 absent (Character 14 Britz and Conway, 2009)   Character 15 of Britz and Conway (2009)

Condition in Paedocyprisa

Features found only in Danionella (from Britz and Conway, 2009) 59 Males with additional flanges to Paedocypris on the os suspensorium confluent with the later process on the second vertebra 60 Males with cartilaginous nodule associated with the anterior face of rib 5 and on the sides of the anterolateral curvature of the anterior swimbladder compartment 61 Males with anterior shift of the genital opening and anus 62 In both sexes, maxillo-mandibulary cartilage between the coranoid process of the lower jaw and the postero-ventral tip of maxilla

 

Apomorphy for Danionella

True

Apomorphy for Danionella

True True

Apomorphy for Danionella Apomorphy for Danionella

True True True True True True

Apomorphy Apomorphy Apomorphy Apomorphy Apomorphy Apomorphy

for for for for for for

Sundadanio Sundadanio Sundadanio Sundadanio Sundadanio Sundadanio

Character from Cavender and Coburn (1992). a Numbers in brackets are character numbers used by Fink and Fink (1981) and referenced by Britz and Conway (2009). Characters identified by Fink and Fink (1981) are all unique and shared-derived characters for Ostariophysi and Order Cypriniformes. Cypriniform synapomorphies identified by Fink and Fink (1981) as homoplasious among Ostariophysi. ** Character numbers from Fink and Fink (1981) are in brackets. Further derived conditions within Cypriniformes as observed in Paedocypris and proposed by Britz and Conway (2009: 402–403). § Characters from Siebert (1987).    Skeletal characters from Britz and Conway (2009:403) for Paedocypris (herein Paedocyprididae) ‘‘that have a derived character state relative to the last common ancestor of cyprinids and that are not affected by developmental truncation, characters, which we like to call progressive”. *

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Features found only in Sundadanio (from Britz and Conway, 2009) 63 Males with co-ossified scapulo-coracoid 64 Males with a serrate dorsal flange on pectoral fin ray 5 65 Males with a large posterior flange on cleithrum 66 Males with a hypertrophied outer arm of the os suspensorium 67 Males with an enlarged rib 5 68 Males with an enlarged, likely drumming, muscle located between the os suspensorium and rib 5

True

161

162

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morphological data supporting the preferred hypothesis; these data and interpretations are presented below. 3.3. Phylogenetic relationships based on morphological characters Apomorphic anatomical characters presented by Fink and Fink (1981), Siebert (1987), and Britz and Conway (2009) for either Cypriniformes, Cyprinidae, or groupings of miniature species, and discussed by Britz and Conway (2009) as to the phylogenetic placement of Paedocypris, can be reinterpreted within the context of the new molecular phylogenetic hypothesis (Fig. 2), as compared to the alternative hypotheses offered by Rüber et al. (2007) and Britz and Conway (2009) using characters presented in Fink and Fink (1981), Siebert (1987), and Britz and Conway (2009) (Table 2, Fig. 3). Morphological character distributions, interpretations, and reinterpretations are presented below and in Table 2 and Fig. 3. 3.4. Reevaluation of morphological evidence for relationships of Paedocypris In their discussion of the evolution of miniaturization, Britz and Conway (2009) examined the evolutionary placement of Paedocypris in light of its possession of some but not all characteristics of the order Cypriniformes and/or their family Cyprinidae, and explained the absence of particular synapomorphies as a consequence of developmental truncation (Fig. 4, ontogeny 3 and not ontogenies 1 or 2). ‘‘The weight of the aforementioned characters indicates that Paedocypris is a member of the Cypriniformes. Within this group it belongs to the Cyprinidae because it lacks the derived characters of the other cypriniform families. Monophyly of the Cyprinidae currently has little support, and its subfamilies are also poorly characterized by shared-derived characters (see Howes, 1991; Cavender and Coburn, 1992). Of Siebert’s, 1987 four derived characters to define the Cyprinidae, Paedocypris only has two (absence of an uncinate process on EB1 and 2; absence of PB1), and of the additional six characters listed by Cavender and Coburn (1992) as potential synapomorphies of Cyprinidae, Paedocypris has none. Of these eight potential cyprinid synapomorphies that Paedocypris lacks, three are inapplicable because the structures in question are absent (interorbital septum formed both by the orbitosphenoid and parasphenoid – interorbital septum absent in Paedocypris; loss of contact between infraorbital 5 and supraorbital – infraorbital 5 absent in Paedocypris; presence of an opercular canal – sensory canals absent in Paedocypris); and lack of the remaining five might be due to the developmental truncation (deep, well-developed subtemporal fossae; anterior opening of the trigemino-facial chamber positioned between the prootic and the pterosphenoid; ossified preethmoids; fused second and third Weberian vertebrae; overlap of PB2 by PB3), and of which one certainly shows a highly derived state (PB2 and 3 at the same level and confluent with each other and EB4 in Paedocypris). However, Paedocypris does possesses a masticatory plate (sensu, Howes, 1981) on the basioccipital process (Fig. 3B and C), a derived feature present in all members of the Cyprinidae (that we have been able to examine) and absent in basal members of other cypriniform families.” (Britz and Conway, 2009: 402–403, emphasis added). The argument that Paedocypris belongs to the Cyprinidae because it lacks the synapomorphies of the other clades within Cypriniformes is neither a logical nor sound phylogenetic argument. The logic of placing a taxon in a group for which it lacks any synapomorphies only because it lacks the synapomorphies of other groups in the clade has historically been used to support unnatural (paraphyletic or polyphyletic groups); perhaps the

absense of synapomorphies is the result of some other process. As addressed elsewhere herein, we propose that the placement of Paedocypris in Cypriniformes is problematic because it forms the sister group to what has previously been known as Cypriniformes. One of the most notable synapomorphies for basal Cypriniformes (as historically perceived) is the bony basioccipital process, a structure that the authors acknowledge that Paedocypris possesses, considers a derived feature of Cyprinidae (historically conceived), and absent from basal members of other cypriniform families. To the contrary, all members of the Cyprinoidea and the Catostomidae have a well-developed posteriorly directed and ossified basioccipital process; the basioccipital process is present in all other Cobitoidea but much reduced in size. Paedocypris, contrary to Britz and Conway (2009) does possess a basioccipital but does not possess a posterior process, displaying the plesiomorphic condition (see their Fig. 3A–E and Fig. 5A–B). The sister-group relationships of any species are necessarily subject to the taxa included in any such analysis and limiting the sampling of species of a group will obviously and significantly influence the outcome, as well as evolutionary conclusions derived from such a phylogeny. In reconstructing the phylogenetic hypothesis for the placement of Paedocypris, the authors neglected to provide any type of matrix of character data for homologies of morphological characters, metrics as evidence of any type of phylogenetic analysis was conducted, or that their hypotheses of homology had been corroborated or falsified; thus, one cannot replicate their study to evaluate the evidence supporting their multiple hypothses. Rather, the unorthodox method of determining sister-group relationships and testing of synapomorphies was justified as follows: ‘‘To develop a hypothesis about the phylogenetic position of Paedocypris among cyprinids, we have chosen the following approach. Rather than comparing Paedocypris with hundreds of representatives of all cyprinid subfamilies in a big parsimony analysis, we decided to focus on other miniaturized cyprinid taxa that we think are more likely candidates as close relatives. This approach follows the rationale that miniaturization has been a gradual process, in which case the closest relative of Paedocypris is expected to be another miniature or very small cyprinid.” (Britz and Conway, 2009: 403, emphasis added). Their study included specimens of other miniature cyprinids, including species of Boraras, Danionella, Horadandia, Sawbwa, and Rasbora, and Danio erythromicron, D. margaritatus, D. rerio, Microrasbora kubotai, M. rubescens, M. nana, Rasboroides vaterifloris, Tanichthys micagemmae, and Trigonostigma hengeli. While the taxa chosen in the analysis was on the basis of other species being miniaturized, their assumption in this instance was also reinforced by the results of Rüber et al. (2007) wherein molecular analyses of cytochrome b sequences alone identified Paedocypris as sister to another genus containing miniature species, genus Sundadanio. Regardless, of these prior findings, the morphological phylogenetic conclusions were inherently biased by the selective sampling of species that were miniatures and the assumption that the closest relative to Paedocypris is expected to be another miniature or very small cyprinid. In such a case, presupposing the range of possible sister-taxa radically influences any alternative relationships or outcomes for Paedocypris; alternative hypotheses for relationships among other Cypriniformes were extremely limited to impossible. Reconstructing evolutionary relationships using anatomical or developmental characters in species with demonstrable developmental ‘‘anomalies” (e.g., truncation, progenesis, etc.) is complicated because it is difficult to determine how the characters used in such an analysis may have been modified by factors other than descent, such as alterations in ontogeny (terminal or non-terminal

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163

Fig. 3. Hypothesized morphological synapomorphies discussed in text and optimized on a simplified tree depicting our current understanding of phylogenetic relationships of the major lineages of the Cypriniformes, including the current hypothesis of relationships for Paedocypris (Paedocyprididae) as the sister group to the Cyprinoidea plus Cobitoidea, relationships of the subfamilies elevated herein and in Chen and Mayden (2009) to families, Danionella within the Rasborine Clade, and Sundadanio in the new family Sundadanionidae. Sister-group relationships within the Cyprinoidea plus Cobitoidea are derived from this analysis, Chen and Mayden (2009), and Chen et al. (2009). Tree contains numbered morphological characters that are discussed in detail in the text and Table 2 herein and are from Fink and Fink (1981), Britz and Conway (2009), and Mayden (unpubl.).

additions and deletions) or environmental conditions that produce morphologies that are not heritable; hence, conflating homoplasy with homology. Conflation of these two different and distinct processes will interfere with accurate identification of synapomorphies. Due to complications of identifying synapomorphies of developmentally truncated taxa, Britz and Conway (2009) argued that the best methods for character comparisons was to follow the logic and methods of Johnson and Brothers (1993) who hypothesized relationships of Schindleria, another highly developmentally-modified teleost. Thus, they chose presumed closely related but non-miniature relatives of Paedocypris and argued that

they could ‘‘identify synapomorphies in earlier developmental stages (of non-miniature taxa), which means comparing the ontogeny of the unminiaturized taxa with the adult stage of the miniaturized taxon.” Johnson and Brothers (1993) used this methodology and logic and ‘‘the osteology of Schindleria and gobioids, with particular emphasis on early developmental stages” to hypothesize synapomorphies for the species. While this method is a potential solution for hypothesizing relationships wherein unusual modifications of morphology or development exists, this logic has the potential to recover sharedcommon ancestry but only if three conditions are met. First, the

164

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Ontogeny 1. Development in the ancestral Cypriniformes species for a Character Transformation Series; letters are developmental character states in ontogeny. Condition E is considered a terminal addition to the Character Transformation Series in Ontogeny 2 (below).

A

B

C

D

E

Ontogeny 2. Developmental in a species prior to the ancestral species of Cypriniformes for the same Character Transformation Series as in Ontogeny 1.

A

B

C

D

Ontogeny 3. Development in a species for the same Character Transformation Series as in Ontogeny 1 that is embedded within the Cypriniformes well after the evolution of the ancestral species to the Cypriniformes but one that has lost condition E. The absence of condition E is considered a terminal deletion and apomorphy for the taxon. This apomorphic condition cannot be distinguished from Ontogeny 2 except through phylogeny reconstruction using other characters.

A

B

C

Identical

D

Fig. 4. Examples of simple ontogenetic linear pathways in the development of morphologies illustrating how an ontology lacking a particular terminal morphological condition (e.g., E) can be confused with an ontogeny that has lost the terminal condition via terminal deletion or truncation. Differentiation of these two radically different hypotheses requires dense taxon sampling of closely related species, all with relevant ontogenies, and character optimization.

ontogeny of the unminiaturized taxa examined must remain intact, without any alterations (deletions or additions) in the ontology it shares with the immediate common ancestor(s) purportedly shared with any purportedly miniaturized taxon descended from that ancestor. Second, no unminiaturized taxa examined will have autapomorphic ontological changes or morphologies as adults. Third, the resultant adult morphology of Paedocypris and other miniatures has strictly been the result of terminal deletions in ontogenetic stages. With any other alterations in the ontology of the taxa their proposed solution would yield misleading results because the adult morphology of a miniaturized taxon will not be one in a series of developmental stages that may be observed in the ontogenetic transformation of the unminiaturized taxa. Likewise, any non-terminal deletions in non-miniature and miniature species would be inconsistent with the developmental truncation hypothesis (Fig. 4). Using these taxa and character information already known for the Ostariophysi and Cypriniformes (from Fink and Fink, 1981), Britz and Conway (2009) hypothesized that Paedocypris should be placed in groups with other miniature species, and in the family Cyprinidae; however, character anomalies existed for Paedocypris relative to the apomorphies of Cypriniformes and Cyprinidae for this taxon (see above). In fact, Paedocypris possesses only four of the synapomorphies for Cypriniformes as outlined by Fink and Fink (1981) (Table 2). The placement of Paedocypris in the family Cyprinidae rested on its lack of apomorphies of any of the other families in the order Cypriniformes, even though the species lacked two of the four previously hypothesized synapomorphies for Cyprinidae, as identified by Siebert (1987), and all of the synapomorphies for Cyprinidae as identified by Cavender and Coburn (1992) (see Table 2 herein for full listing of characters). A review of the morphology of Paedocypris presented in Britz and Conway (2009), Fink and Fink (1981), and Cavender and Coburn (1992) and the hypothesized synapomorphies for Cypriniformes and groupings within the order, reveals that the characters

examined and discussed by Britz and Conway (2009) relevant to the placement of Paedocypris within the Rasborine Clade have alternative interpretations when the character state distributions are viewed in a broader context for the Cypriniformes and Otophysi. Many of those possessed by Paedocypris are consistent with its placement as the sister group to Cypriniformes, as the order was traditionally conceived prior to the discovery of Paedocypris. These reinterpretations of character arguments, supporting the sistergroup relationship of Paedocypris (Paedocyprididae, Paedocypridoidea) are consistent with the results presented herein based on a robust molecular data set and analysis. Before reviewing and discussing the morphological characters relevant to the placement of Paedocypris, there are three fundamental considerations that we contemplated in this evaluation. The first was basic outgroup evaluation but at a broader context than the miniature species and select members of the Rasborine Clade. The second involved interpretation of the characters used and the logic behind the absence of an apomorphic character for Cypriniformes or Cyprinidae based on the assumption of terminal deletions. The third involved the interpretation of an array of character states being lost in Paedocypris due to terminal deletions in development versus the character state never being present in Paedocypris and thus masquerading as a terminal deletion in a species that never had it as part of its developmental trajectory. Statements of alterations of developmental sequences, whether additions or deletions or terminal or non-terminal modifications, require a fairly robust and dense taxon sampling to determine which of these possibilities are the most parsimonious conditions. The taxon sampling in the analysis by Britz and Conway (2009) and their unknown methods of phylogeny reconstruction preclude the differentiation of any of these developmental alternatives. What may appear as a developmental truncation or loss of one or more terminal conditions in a developmental pathway in a taxon may be equally or more parsimoniously interpreted as a taxon that never possessed the terminal character state (Fig. 4). This is the

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case for some of the character transformations and character states identified for Paedocypris by Britz and Conway (2009). The morphological character arguments for Paedocypris forming the sister group to all Cypriniformes (as traditionally viewed before the discovery of Paedocypris) are presented below. These characters are, in some instances, reinterpreted from the view of Britz and Conway (2009) but are consistent with the placement of Paedocypris based on the molecular evidence presented herein and the hypothesis that miniaturization has evolved multiple times in the history of this order. From the sampling of taxa used in this analysis the number of miniaturization events is indicated with an  in Fig. 2. As to the number of times that miniaturization occurred within the order, this conclusion will have to await a more thorough sampling of taxa, especially within the Rasborine Clade, to more clearly refine sister-species relationships for the optimization of the evolution of miniaturization. Deciphering the number of ways that a miniaturized phenotypic condition of species has been achieved must follow this type of optimization with detailed anatomical, and perhaps more detailed investigations via Danio rerio and existing mutant phenotypic and genotypic databases and an anatomical ontology (see discussion below). The morphological characters discussed by Britz and Conway (2009) and additional characters provided herein are listed in Table 2 and are associated with particular hypotheses as synapomorphy, autapomorphy, or parallelism for a particular taxon. The distributions of these characters, using the same character numbering scheme as in the Table 2, are depicted in the summary phylogeny of major groups of Cypriniformes (Fig. 3). The monophyly of the Otophysi is not a matter of contention here and the synapomorphic condition of the Weberian apparatus as described by Fink and Fink (1981) is taken as support of this clade. With regard to characters hypothesized to support the monophyly of Cypriniformes (inclusive of Paedocypris), we identify 14 characters. Characters 3–5 and 11–17 have all been proposed as synapomorphies for the Order Cypriniformes and we agree with this interpretation. For Characters 2, 6–10, and 18 we differ in character interpretation from both Fink and Fink (1981) and Britz and Conway (2009) and these are discussed below and illustrated in Figs. 3 and 5. Character 2. Bony Kinethmoid. The presence of a kinethmoid is interpreted as a synapomorphy for Cypriniformes (inclusive of Paedocypris) (Fig. 3; 2A), however, the ossification of this element is considered a further derived state (Fig. 3; 2B) uniting all Cypriniformes, exclusive of Paedocypris. The cartilaginous condition typical of Paedocypris is considered a precursor to the ossification and under this interpretation serves as the synapomorphy of Cypriniformes (Table 2, 2A). Character 6. The preethmoid is interpreted to have evolved in the ancestor to all Cypriniformes, exclusive of Paedocypris (6A). Character 7. The palatine of Cypriniformes possesses a process that abuts against the mesethmoid (Figs. 3 and 5D and E – PP and PAL herein). In all Cypriniformes, except Paedocypris, this process is well developed and its enlargement is considered a synapmorphy of this group (7B). The small ‘‘nubbin” of the process in Paedocypris is interpreted as not only the first occurrence of the structure in the evolution of the Cypriniformes but the precursor to the larger process in all other taxa (7A). Character 8. The lack of a palatine-endopterygoid articulatory facet is interpreted as plesiomorphic for the order and a condition retained in Paedocypris. The articulation facet (Figs. 3 and 5D and E – ENP and PAL herein) evolved in the ancestor of Cobitoidea plus Cyprinioidea and is a synapomorphy of this group (8A). Character 10. Paedocypris lacks an ascending medial process of the premaxilla. This process was identified by Fink and Fink (1981) as a synapomorphy of Cypriniformes (10B), a character interpretation with which we concur. However, the small expansion of the premaxillary in Paedocypris is hypothesized as a synapo-

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morphic precursor (10A) to the extended maxillary process (10B); the ascending process thus represents a synapomorphy for Cyprinoidea plus Cobitoidae (10B), to the exclusion of Paedocypridoidea. Character 18. Fink and Fink (1981) hypothesized that the presence of maxillary barbels was a synapomorphy for Cypriniformes. However, in examination of the distribution of maxillary barbels in the order, they are present in all of the major lineages in Cyprinoidea but are absent in Catostomidae and Gyrinocheilidae, two basal, non-sister group lineages of Cobitoidea. Thus, it is hypothesized herein that the absence of barbels is a plesiomorphic condition for Cypriniformes and they evolved independently in the ancestral Cyprinoidea (18A*) and in the ancestor to all cobitoids less catostomids and gyrinocheilids (18A*) (as well as many other groups of fishes). The lack of barbels in Paedocypis is interpreted as a plesiomorphic absence. The barbels are quite different across the Cypriniformes and a detailed survey of the anatomical structures and distributions of barbels in the order is warranted and may reveal additional insights into the evolution of this character complex and relationships among groups. Character 19. The masticatory plate, present on the ventral surface of the basioccipital (Figs. 3 and 5H – PPD herein) is interpreted as a synapomorphy for the Cypriniformes (19A), including Paedocypris, but the plate is heavily modified in many species of Catostomidae as a palatal organ (19B). This plate is present in a reduced size in Gyrinocheilidae, some Catostomidae, but is presumably lost other cobitoids (19B), the latter being an observation worthy of much more detailed investigation. Character 20. In Cypriniformes the pharyngeal process of the basioccipital is an ossified, posteriorly-directed process that is largest in Cyprinoidea (Figs. 3 and 5G, H, and J – PPBO) and the basalmost members of the Cobitoidea, Family Catostomidae, and is usually reduced in size in other Cobitoidea. Britz and Conway (2009) refer to a basioccipital process in Paedocypris but the species lacks this posterior process to the basioccipital (Britz and Conway, 2009; fig. 5 vs. Devario cf. malabaricus, PhPr in fig. 14. Paedocypris lacks this process entirely as do outgroups and this condition is considered plesiomorphic. Thus, the presence of the pharyngeal process of the basioccipital is hypothesized to be a synapomorphy for Cobitoidae plus Cyprinoidea (30A). With the reinterpretation of the above characters there remains two morphological characteristics that are synapomorphic for the Cobitoidea plus Cyprinoidae. These include Characters 6, 8, and 20. Character 20 is discussed above. Characters 6 and 8 include the presence of a preethmoid and the palatine/endopterygoid articulatory facet, respectively (Table 2, Figs. 3 and 5). A total of 10 characters have been hypothesized to be synapomorphic of Cyprinoidea by various authors (Siebert, 1987; Cavender and Coburn, 1992; Britz and Conway, 2009). We differ in the interpretation of eight of these characters relative to the placement of Paedocypris in Cypriniformes. For Characters 24 and 25 we concur with the interpretation that these are autapomorphic conditions for Paedocypris, possibly developed by a developmental terminal deletion; at present we are neutral with Britz and Conway’s (2009) interpretation of Character 30 and consider this an autapomorphy of the taxon. Character 21. The lack of the uncinate processes on EB1 and EB2 is considered a synapomorphy for Cyprinidae by Fink and Fink (1981). Given that the historic taxonomic composition of Cyprinidae has changed dramatically in recent years with the recognition of multiple families in what was once only Cyprinidae (Chen and Mayden, 2009; herein) and the superfamily will likely be dissected further as more phylogenetic information on these fishes is developed, the placement of this character is better referred to as a synapomorphy for the Cyprinoidea, a well-supported monophyletic group in every morphological and molecular analysis to date. Paedocypris lacks the uncinate processes on EB1 and EB2, a condition

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Fig. 5. Morphological structures and particular characters of Cypriniformes discussed in text. Illustrations modified from Mayden (1989). (A) Dorsal view of cranium of cyprinoid. (B) Lateral view of ethmoid complex. (C) Ventral view of ethmoid complex and vomer. (D) Dorsal view of palatine articulating in facet of endopterygoid; palatine also displays enlarged dorsally directed process that connects to kinethmoid. (E) Lateral view of pterygoid series and palatine, illustrating relationships of the endo- and ectopterygoids, articular facet for palatine in endopterygoid, and dorsal stem of palatine connecting to kinethmoid. (F) Lateral view of interorbital septum illustrating formation by orbitosphenoid and parasphenoid and lateral commisure of the prootic with the facial foramen exiting at junction of prootic and pterosphenoid. (G and H) Lateral and ventral views, respectively, of enlarged pharyngeal process of basioccipital typical of Cyprinoidea and basal Cobitoidea and masticatory plate or the pharyngeal pad area (PPD). (I) Ventrolateral view of cranium showing pterotic (also in K and L below) containing the trigemino-facial chamber and the anterior opening (FVII) between prootic and pterosphenoid. (J) Posterior view of cranium illustrating the subtemporal fossa and the pharyngeal process of the basiocciptial complex. (K and L) Enlarged views of prootics of two species of Cyprinella (Leuciscidae) showing how the trigemino-facial chamber (right side of each) can have the anterior opening (FVII) either at anterior margin and shared with pterosphenoid or completely contained in the prootic. VO, Vomer; FR, Frontal; PE, Preethmoid; LE, Lateral ethmoid; SE, Supraethmoid; N, Nasal; SPH, Sphenotic; EPB, Epiphsyseal bar; DF, Dilator fossa; SO, Supraorbital; PA, Parietal; DPT, Dermopterotic; PTF, Post-temporal fossa; EOC, Exoccipital; LOC, Lateral occipital foramen; SO, Supraoccipital; EPO, Epiotic; OF, Olfactory foramen; ME, Mesethmoid; OSP, Orbitosphenoid; PS, Parasphenoid; ENP, Endopterygoid; MP, Median process of palatine; MPT, Metapterygoid; ECT, Ectopterygoid; QA, Quadrate; V1, First vertebra; PPBO, Pharyngeal process of basioccipital; BO, Basioccipital; PPD, Pharyngeal pad; APT, Autopterotic; STF, Subtemporal fossa; PTS, Pterosphenoid; PRO, Prootic; FIC, Carotid foramen; FVII, Facial foramen; FM, Foramen magnum; IC, Cavum sinus impar. Horizontal bars represent 1 mm.

considered herein to be plesiomorphic. Thus, the presence of uncinate processes on EB1 and EB2 is a synapomorphy of Cyprinoidea (21A) to the exclusion of Paedocypris.

Character 22. Presence of PB1 is hypothesized to be a synapomorphy of Cyprinoidea (22A). Paedocypris lacks PB1 and similar logic discussed in Character 21 applies here.

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Character 23. The presence of an interorbital septum formed by the orbitosphenoid and parasphenoid has been hypothesized to serve as a synapomorphy of Cyprinoidea. However, this character is more widespread than previously reported and is found in many species of Cobitoidea and Cyprinoidea. The interorbital septum can be restricted to only the orbitosphenoid in some Cyprinoidea (Figs. 3 and 5B, F, and I – ORS, herein). Thus the presence of an interorbital septum should be considered a synapomorphy of the Cyprinoidea plus Cobitoidea (23A). The absence of the septum in Paedocypris is thus considered a plesiomorphic condition, placing this taxon outside of the Cyprinoidae. However, in the illustration by Britz and Conway (2009; Fig. 3B) the interorbital septum, being small and formed by the orbitosphenoid as in some Cobitoidea and Cyprinoidea, may be considered an apomorphic precursor to the more well-developed condition involving both the orbitosphenoid and parasphenoid in the combined Cyprinoidea and Cobitoidea. Character 26. A deep, well-developed subtemporal fossa (Figs. 3 and 5A and J – PTF herein) has been hypothesized to be a synapomorphy of the Cyprinoidea (26A), an interpreation that we continue to support. Paedocypris lacks a post-temporal fossa, a condition hypothesized by Britz and Conway (2009) to represent a developmental truncation. It is difficult to image how this fossa could be absent due to a developmental truncation, a consideration also pondered by the former authors. We hypothesize that the lack of the post-temporal fossa is a plesiomorphic condition shared with Cobitoidea. Character 27. The anterior opening of the trigemino-facial chamber, positioned between the prootic and pterosphenod (Fig. 5I, K, and L – FVII, herein) has been hypothesized as a synapomorphy for the Cyprinoidea, an interpretation with which we concur. Paedocypris lacks this anterior opening, and as in Character 26, is interpreted by Britz and Conway (2009) as an absence due to developmental truncation. We argue that the presence of this anterior opening is a synapomorphy for Cyprinoidea and using the logic provided in the discussion of Character 26, the absence of this opening, as in Paedocypris, is a plesiomorphic condition. Character 28. The fusion of the second and third vertebra has been hypothesized as a synapomorphy for Cyprinoidae (28A). The absence of this synapomorphy in Paedocypris, as in characters listed above, was interpreted Britz and Conway (2009) as a result of developmental truncation. We disagree with this interpretation and consider the absence of this fusion to be a plesiomorphic condition in Paedocypris employing parsimony logic outlined in Characters 26 and 27, thus excluding it from the Cyprinoidae. Character 29. The overlap of PB2 and PB3 has been hypothesized to be a synapomorphy of the Cyprinoidea (29A), an interpretation that we continue to support. The lack of overlap of these two elements in Paedocypris is interpreted by Britz and Conway (2009) as a result of developmental truncation. We argue that the explanation requiring the fewest ad hoc explanations is that the condition has simply never been present in Paedocypris and it possesses the plesiomorphic condition, thus excluding it from Cyprinoidea. Character 30. While we tentatively agree with the interpretation of PB2 and 3 being at the same level and confluent with each other and EB4, as described by Britz and Conway (2009) it is likely an autapomorphy for Paedocypris, Paedocyprididae, and Paedocypridoidea; determination of this must await a more detailed survey of the anatomy of the Cypriniformes. In addition to Characters 9, 17 (further derived condition of loss of post-cleithrum), 24, and 25, we consider Characters 31–35 to be apomorphic for the clade inclusive of species of Paedocypris, or the Paedocyprididae or Paedocypridoidea as proposed herein. Some characters were hypothesized by Britz and Conway (2009) to be synapomorphies for a Paedocypris plus Danionella lineage (36–41); however, given the lack of support for the characters discussed above placing Paedocypris within the Cyprinoidea these

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characters are interpreted herein as independently derived in each of these taxa. Character 15 of Britz and Conway (2009) could not be located in their paper. The remaining characters identified in Table 2 are either proposed synapomorphies uniting Paedocypris, Danionella, and Sundadanio, or are elements lost in these three taxa. Our argument for the lack of evidence for the placement of Paedocypris in Cyprinoidea, as well as the strong internal lineage support for Danionella and Sundadanio being more closely related to two different highly divergent and non-sister group lineages, based on both morphological and molecular character variation, necessitates these to be independently derived in each of these three taxa, likely through what Britz and Conway (2009) determine to be developmental truncation or convergence. However, once a more thorough morphological examination of more species of the rasborins has been accomplished, some of these characters may be found to be phylogenetically informative relative to Danionella and possible close relatives, possibly even species with hypothesized developmental truncation. As there is no molecular evidence for the sister-group relationships of Sundadanio with Danionella and either of these with Paedocypris, the similarities observed between these taxa are considered to be homoplasious. While strong evidence exists for the new phylogenetic placement of the smallest vertebrate species of the genus Paedocypris as the sister group to the most diverse clade of freshwater fishes, and not a member of a clade containing other miniature fishes, attempts to theorize more broadly as to the patterns and processes associated with the evolution of miniaturization and the impact of body size evolution on diversification in this diverse order would be premature without more complete taxon sampling. However, the diversity of size, distributional patterns, and ecologies of these fishes evaluated in a phylogenetic context, as advocated by McClain and Boyer (2009), can be realized in the near future for the order, and patterns of diversification relative to an array of ‘‘niche” related biological attributes for these species relative to body size can be evaluated using geographic-, time-, and phylogeneticcontrols. Accompanying the growing consistency for phylogenetic evidence of relationships of species in the Cypriniformes has come the consistent support of major clades within the former family Cyprinidae that are now recognized as separate families (Chen and Mayden, 2009; herein), rendering the former Cyprinidae equivalent to Cyprinoidea. The revised family Cyprinidae includes species of the former subfamily Cyprininae and this clade is sister to the family Psilorhynchidae. Herein, three former subfamilies of Cyprinidae, Acheilognathinae, Leuciscinae, and Gobioninae, are elevated to family status and in keeping with consistency between phylogenetic relationships and a natural classification we elevate the available names for families Leptobarbidae Bleeker 1863a– 1864 and Tincidae Kryzanovsky 1947, and describe the new families Tanichthyidae and Sundadanionidae below (Fig. 2). With Paedocypris forming the sister group to the clade Cyprinoidea plus Cobitoidea (Figs. 2 and 3) the classification of this species in the former family Cyprinidae renders that family as an unnatural polyphyletic group; even considering the revised classification of Cyprinidae its placement in the newly composed Cyprinidae is inappropriate. Thus, as part of our continuing reevaluation of the classification of Cypriniformes Paedocypris is now recognized in a new superfamily and family diagnosed below.

4. Discussion The order Cypriniformes is the most diverse clade of freshwater fishes (Nelson, 2006; Mayden et al., 2007, 2008), containing species and clades that display an amazing diversity of morphologies,

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natural histories, and body sizes (Winfield and Nelson, 1991; Nelson, 2006; Froese and Pauly, 2009). This diversity and their ease of care and propagation, combined with the rapidly growing knowledge of the phylogenetic relationships of these fishes (Mayden et al., 2007, 2008; Chen and Mayden, 2009) makes this clade particularly appropriate as a model clade of freshwater fishes that can be found on nearly every continent and appropriate for detailed investigations of the evolutionary and coevolutionary origins of attributes, behaviors, and biogeographies within a phylogenetic context. Through the emergence of the phylogenetic paradigm many years of research have demonstrated the necessity of sister-group relationships as an historical framework for addressing any type of evolutionary question at any level from the individual organism to supraspecific taxa (Mayden, 1992). Thus, the phylogenetic placement of the world’s smallest vertebrate is of great importance as ongoing and future studies will be examining this species and presumed close relatives for evolutionary inferences as to the origin of this clade of miniature fishes and other clades of miniature species within the order. Assuming that all miniature fishes in Cypriniformes are closely related and a sister-group relationship must be within this subset of taxa is not a valid assumption, a conclusion clearly demonstrated herein with multiple nuclear genes. Efforts to investigate the evolutionary mechanisms or processes responsible for the origin of miniaturization or its possible maintenance in specific types of habitats would have been grossly mislead by a selective sampling of taxa for the search for the sister group to Paedocypris and other miniature cyprinids. 4.1. What is the sister-group relationship of the smallest vertebrate species? Rüber et al. (2007), using sequences of cytochrome b, identified the sister group to Paedocypris as another group of miniature rasborine cyprinids from southeast Asia, genus Sundadanio. They recognized at least 21 species of cyprinids from nine genera in South and Southeast Asia and 12 species in three genera from Africa as also evolving through some type of process ultimately resulting in miniaturization. In their analysis of Paedocypris, the authors hypothesized that miniaturization occurred multiple times, although their resolution of the subgroup within their Clade D, the Rasborin Clade, was poorly resolved; however, nearly all of the evolutionary occurrences were found within one particular clade, the ‘‘Rasborin Clade” (see Rüber et al., 2007, Figs. 1–3, herein). Accurate estimates of ancestral character state reconstruction for the evolution of miniaturization depends upon at least three important factors; an accurate phylogenetic inference, sampling of all or nearly all extant taxa, and unambiguous delineation of the quality referred to as ‘‘miniaturization.” What exactly is miniaturization? Can it be more clearly defined other than a body size less than a set number of millimeters? If so, then perhaps as in hypotheses of character homology one may determine that two structures appearing the same at one scale do not appear to be the same at another and should be coded as non-homologous traits or homoplasies. The same is likely true of miniaturization. A reduced body size could have been arrived at in a variety of ways, only some of which involve terminal deletions. Therefore, clearly defining miniaturization is a critical first step. Both the analysis by Rüber et al. (2007) and Britz and Conway (2009), as well as the analysis herein includes a number of species identified as miniatures but all studies suffer from having incomplete taxon sampling for the order Cypriniformes, a sampling problem that can dramatically alter ancestral-state reconstructions and hence isolating evolutionary occurrences and events associated with or responsible for miniaturization. However, the three studies are not comparable as to the weight of evidence supporting the sister-group relationships of Paedocypris. Earlier we addressed the shortcomings of the two

previous phylogenetic studies and invalid assumptions of taxon sampling and character homology by Britz and Conway (2009) and the problems associated with using only cytochrome b sequences for this level of question. Previous molecular phylogenetic studies for Sundadanio and Danionella, as well as other miniature cyprinids, have been conducted using multiple genes and genes that are not as readily influenced by saturation levels at this level of analysis as is cytochrome b. In none of these studies have the miniature cyprinids or even Danionella and Sundadanio ever been identified as closely related, as indicated by Britz and Conway (2009) and to some degree by Rüber et al. (2007). In parsimony analyses, Mayden et al. (2007), using two nuclear and five mitochondrial genes found Danionella sister to Opsaridium and this clade sister to Sundadanio; however, the support for and within this clade was poor. In the same paper the ML tree identified Sundadanio as the basal sister group to a large apical clade wherein Opsaridium formed the sister group to two major clades. One clade included Danionella sister to a monophyletic group including species of Chela, Microrasbora, Inlecypris, Devario, and Danio, mostly containing non-miniature species. The sister clade included Esomus as the basal sister group to a clade of species of Rasbora with Trigonostigma (= Rasbora) and Boraras (= Rasbora) embedded within this clade; again, a group of cyprinids containing few small or miniature taxa. Using variation in exon 3 of Rag 1 and increased taxon sampling (49–117 taxa), Mayden et al. (2008), identified Sunadanio as sister to Esomus with very high support from Bayesian analysis and this clade in a trichotomy with two other clades; one of these clades was composed of Danionella as the basal sister group to a clade including Danio, Chela, Microrasbora, Devario, and Inlecypris, again with limited small or miniature species. The third clade included Trigonostigma and Horadandia (= Rasbora) mixed in with Rasbora. Analyses by Conway et al. (2008), using sequence variation from Rag 1, identified Danionella as a monophyletic group sister to a clade inclusive of Danio, Devario, Microdanio, Chela, Microrasbora, and Inlecypris; Sundadanio was sister to Esomus. These two clades were in a polytomy with a third clade inclusive of Rasbora, Trigonostigma, Boraras, and Horadandia (all forming Rasbora). Analyses by Mayden et al. (2009) and Chen and Mayden (2009) did not have representation of Danionella, Sundadanio, or both and none of the above studies included Paedocypris. Clear conclusions consistent across these studies, however, are that species of Danionella and Sundadanio are more closely related to species of other genera than they are to one another and that Trigonostigma, Boraras, and Horadandia should be synonymized with Rasbora. In our current study we included several of the previously identified miniature cyprinids and miniaturization evolved, depending upon ancestral character state reconstructions in some portions of the tree, at least nine times (Fig. 2; taxa identified by ), inclusive of an interpretation of an independent origin in Paedocypris. In our reconstructions with Paedocypris as either the sister group to all Cypriniformes (preferred hypothesis, Fig. 2, Table 1) and the alternative, less probable hypothesis as the sister group to all Cyprinoidea (Table 1), an independent origin of miniaturization in Paedocypris may be most parsimonious; however, this latter conclusion ultimately depends upon the sister-group relationships of other Otophysi lineages and basal members of those lineages to be resolved in the future, their body sizes, and the optimization of body size within this evolutionary context. It is possible that the miniature body size of Paedocypris is a retained-primitive attribute and that the ancestral Cypriniformes (Paedocyprididae sister to Cobitoidae plus Cyprinoidea) was a miniature species. Of the Cyprinoidea examined in this study, however, miniature species are restricted to the ‘‘Rasborine Clade” and the family Sundadanionidae. The latter occurrence, given the current taxon sampling, appears to be an independent origin. The number of evolutionary

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events associated with miniaturization in the Rasborine Clade, however, is not as obvious given current taxon sampling; a more concrete number of events must await more thorough taxon sampling of this major group. Miniaturization may have characterized the ancestral species leading to the two sister clades, one containing the basal sister taxon Horadandia atukorali and the other containing the basal sister taxon Esomus logimanus (Fig. 2), with independent reversals occurring in each clade. Other alternative reconstruction can be hypothesized with multiple independent origins in this larger subclade within the Rasborin Clade in either individual species or ancestral species. Unfortunately, until such time that more taxa, miniature or not, are included, especially in the Rasborin Clade as well as taxa near the sister taxa Leptobarbus and Sundadanio, a clear resolution as to the evolution of miniaturization either through developmental truncation or other mechanisms will not be possible. Furthermore, a more clear description of what constitutes the quality of miniature must be more thoroughly investigated and described so that it is clear that researchers are in fact referring to the same quality across the Cypriniformes. These reconstructions, accompanied with detailed developmental studies like those of Cubbage and Mabee (1996), Bird and Mabee (2003), Grande and Young (2006), and Britz and Conway (2009) will be essential in deciphering not only the number of evolutionary occurrences of miniaturization but also whether or not it is achieved in a similar manner in all of these independent occurrences. Based on the current detailed analysis of nuclear gene loci it is evident that previous hypotheses as to the phylogenetic relationships of the miniature species of the formerly recognized family Cyprinidae (Order Cypriniformes), including Paedocypris as the smallest vertebrate species, have all been compromised by either a taxon or character sampling anomaly and miniaturization has occurred multiple times and possibly by different means within the Cypriniformes. This pattern of multiple, independent cases of lineages leading to a miniaturized body size makes the evolution of these species and their close relatives even more intriguing, as parallel evolution of traits (sensu lato) provides outstanding opportunities to seek not only causal mechanisms but the underlying broader conditions wherein which such an ‘‘anomaly” may have arose or would be predicted to arise (Mayden, 1992). Cases of parallel evolution of attributes, when controlled for with phylogenetic relationships, provide optimal opportunities to investigate evolutionary mechanisms and constraints involved, as each instance (ancestral or descendant species) represents a replication of an historical experiment (Mayden, 1992). Thus, the origin of miniaturization within the Cypriniformes is an area of much needed future investigation that can provide valuable insight into not only evolutionary mechanisms and speciation, but may offer extremely valuable information via ongoing research on Danio rerio relative to human-related diseases. With increased inventory work in poorly inventoried areas for new species like the phenomenal find of Paedocypris and once a well resolved phylogeny for most or all of the species of Cypriniformes exists or at least those within the ‘‘Rasborine Clade” then these phylogenetic data may be combined with ongoing research with both morphological ontologies (Mabee, 2006; Mabee et al., 2007a,b; Dahdul et al., 2010a,b; Balhoff et al., 2010) and the knowledgebase available through ZFIN, Danio rerio genetics, morphology, behavior and biology, and the Zebrafish network for breakthroughs into genetic factors controlling body sizes, truncated developmental pathways, as well as environmental conditions that may be responsible for activating or deactivating genetic controls. Kottelat et al. (2006) noted that the majority of miniature fishes in Southeast Asia reside in peat swamp forests that are highly acidic. Given that the water in these forests is more acidic due to decaying plant materials acidity itself may play or may have played an important role in

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either the maintenance of miniature body size or its evolution, respectively. In either case, hypotheses such as these must be tested through collaborative efforts between the phylogenetic and Danio rerio evo–devo communities once a fully resolved phylogeny of the clade is available. 4.2. Cypriniformes as a model clade for research in emerging frontiers for evolutionary biology The evolution of miniaturization is clearly an interesting phenomenon as it is not only very likely linked to many intriguing biological and physiological challenges for these species but it is one extreme to a very broad continuum in the Order Cypriniformes where some species are as large as a human. Given the great diversity of biologies, anatomies, physiologies, behaviors, and ecologies in the order, accompanied by the broad distribution of Cypriniformes fishes across the planet, their availability in nearly any ecosystem, and the continuing focus on the evolution of species in the order, it is proposed that the Cypriniformes be considered and used as a model clade of fishes for detailed evolutionary, developmental, and ecological studies. The refinement of the evolutionary relationships of these fishes through the Cypriniformes Tree of Life Initiative, derived from high taxon sampling and diverse character sets, offers the underlying evolutionary relationships necessary to further expand on this tree of life at finer scales and/or study underlying evolutionary mechanisms of the diverse nature of the group. Furthermore, the most famous model organism for ‘‘fishes” is Danio rerio, Zebrafish, a member of Cypriniformes, and this offers incredibly important opportunities for advancing our understanding on many fronts of comparative and evolutionary biology. Integrating the many detailed studies of morphology, development, behavior, ecology, and genomics and mutant phenotypes and genotypes of Danio rerio with knowledge that researchers gain from other Cypriniformes species for these same types of characteristics offers a new frontier. As an example, many researchers are surveying the anatomy of many cypriniform fishes that possess naturally occurring character state variants or what we may refer to as wild ‘‘mutant” phenotypes. Surveys of these wild mutant phenotypes as systematic traits of cypriniform species and comparisons of this variation to laboratory phenotypic mutants offers our first opportunity to decipher the underlying genetic changes associated with speciation, origins of apomorphic characters, and the minimum amount of genetic change associated with speciation (Fig. 6). Using the documented underlying genetic controls over traits of this nature in detailed studies of Danio rerio with the diversity and variation in diversity in the biologies of species in the order through newly emerging ontologies in anatomy (Mabee et al., 2007a, b; Mayden et al., 2007; Dahdul et al., 2010a,b; Balhoff et al., 2010), behaviors, ecologies, etc. offers an extremely rich future for obtaining direct inferences of evolutionary mechanisms involved in simple to complex organismal attributes. Through the use of ontologies and knowledge bases derived from Danio rerio it is very likely that the near future holds promise to mapping not only apomorphies on species-level trees but also direct evidence for linkages of specific genetic mutations underlying these apomorphies (Fig. 6). In fact, many systematists, taxonomists and evolutionary biologists have recently been exceedingly anxious about the ‘‘overwhelming” appearance of molecular studies being preferred by funding agencies and journals over similar types of studies that could and should be done using morphological data. While we do not agree with the observation by some that morphology is being lost in biodiversity science, we predict that if it is being selected against in these arenas its renaissance and high-levels of funded research will come through the critical need for well-educated and trained morphologists as more and more ontologies like that being developed by the Phenoscape community (Dahdul et al., 2010a, b; Balhoff et al.,

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Fig. 6. Hypothetical example illustrating the idea of linking phenotypic changes (anagenesis on phylogeny) to underlying genetic (genomic) change through developmental biology, genetics, laboratory mutants and wild types of the model organism Danio rerio with ‘‘wild-type” ‘‘mutants” (derived characters) of other Cypriniformes fish species in evolutionary studies. (A) Wild-type (left) and mutant-type (right) phenotypes of mandibles of captive Danio rerio (mutant ‘‘toothed” pattern of mandible is hypothetical in Danio rerio; non-‘‘toothed” mandible pattern is the normal wild-type for Danio rerio). Underlying genetic changes are known for the mutant phenotype. (B) Tracing of actual wild-type ‘‘toothed” pattern present in the species Danionella dracula (derived from Britz et al., 2009, Fig. 2). Underlying genetic changes responsible for producing the hypothetical toothed mutant in Danio rerio (A) is hypothesized be the same genetic architectural change controlling the development of the ‘‘toothed” pattern as seen in Danionella dracula. (C) Hypothetical examples of the wild and mutant phenotypes observed in laboratory Danio rerio for the tips of the pharyngeal teeth on the fifth ceratobranchial arch. The hooked tooth type is the naturally occurring wild-type phenotype. The small, blunt, grinding tooth tip type is the mutant phenotype with known genetic origins derived from genotypic mutations. (D) Hypothetical wild phenotypes observed in other species of Cypriniformes. The enlarged grinding surface of the wild caught species is hypothesized to be the result of mutations to the same genetic architecture producing the small, blunt grinding surface on pharyngeal teeth as in laboratory Danio rerio. (E) Expected outcome available for systematic and evolutionary biology by synthesizing and linking information in examples (A–D) via anatomical ontologies and phenotypic (wild, mutant) data derived from combining studies on Danio rerio with those in systematic biology. Laboratory mutant phenotypic and genotypic data derived from Danio rerio (A–D) where phenotypic mutants have known gene changes or known gene changes produce a series of different mutant phenotypes. In systematic and evolutionary biology ‘‘wild-type” phenotypes can be apomorphies and plesiomorphies. Apomorphies can be displayed on a phylogeny wherein those derived from some type of mutations to the same gene(s) can be colorized identically, providing valuable information as to not only degree of genetic change minimally associated with speciation but also anatomical changes that are not actually independent systematic characters.

2010) and their interfaces with other databases involving morphological and genomic data. Imperative in the success of this inevitable growth in morphology is collaboration and mutual respect for the different disciplines emphasizing different data types in systematics and current morphologists mentoring, with an open mind to alternative data and methods for phylogenetic reconstructions, more students in the discipline. The extreme variation in body size within the Cypriniformes together with the inherent influence of body size on so many different attributes of fishes (Helfman et al., 2009), offers a remarkable opportunity for future detailed investigative and integrative research on evolutionary mechanisms in these fishes. The newly resolved relationships for Paedocypris, one of the most unusual Cypriniformes species, offers a more direct inference into distinguishing between genetic versus underlying environmental influences in its development. 4.3. Is accurate phylogenetic inference even possible with parallel evolution of morphologies? Regardless of the study examining the relationships of Paedocypris, it is clear that miniaturization (as defined by previous authors) has occurred more than once in the Cypriniformes. Accompanying miniaturization appears to be a suite of morphological similarities

(apomorphies, autapomorphies, or plesiomorphies) that have been hypothesized as terminal deletions in development. However, not all of the traits identified by Britz and Conway (2009) regarding relationships of Paedocypris are features that occur at or the near the terminus of development as would be predicted with terminal deletions. These instances should be identifiable as instances separate from terminal deletions and can be separated from other instances where the losses are identical between taxa. Theoretically speaking, accurate phylogenetic reconstructions of relationships of species are possible if the basic assumptions of phylogenetics are met and the analysis benefits from what we have learned from many years of testing sampling methods for molecular phylogenetic studies. One of the most important problems that seems to be plaguing the origin of miniaturization in Cypriniformes is the definition of the condition itself; inherent assumptions are made that all small Cypriniformes are related to one another or the characteristic of being small actually has some meaning in a trajectory of progressive evolution along a lineage. Like molecular studies, accuracy of phylogenetic inferences based on morphological characters is predicted to benefit from increased taxon and character sampling. We predict it highly unlikely that non-sister group miniature Cypriniformes (when a clear definition exists) will be found to resolve as sister taxa if a thorough survey of species is conducted

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and the species are sampled for as many characters as possible from the multiple morphological systems that have historically proven to be useful in so many other studies. These data, combined with known developmental time lines for species, will hopefully reveal accurate inferences of sister-group relationships. However, when the situation of miniaturization is viewed from a distance without thorough sampling of data and with selective sampling of mostly miniature taxa, one cannot be surprised by the resulting sister-group relationships. The origin of a small body size among aquatic organisms holds many intriguing questions as to how they cope with their surroundings, whether it be one or multiple different ways. However, the reconstruction of the evolutionary relationships of species should be possible if the particular situation is approached with greater scrutiny. Very similar questions were recently posed for convergent evolution in the morphologies of cave-adapted salamanders, and results of these authors are in agreement with our hypothesis for resolving relationships and morphological evolution in Paedocypris. In a molecular and morphological systematic study of caveadapted salamanders of the genus Eurycea Wiens et al. (2003) encountered a similar situation as described herein for miniature Cypriniformes. In their analysis the question was raised as to how best to identify cases of convergence and differentiate them from cases of common ancestry when a subset of the taxa happen to occupy perhaps an unusual habitat (or niche) that may lead one to believe that convergence has occurred in morphologies, physiologies, etc. due to the unusual and perhaps highly selective environment (cave dwelling, or in the case of Paedocypris possibly acidic swamp areas associated with miniature cyprinid species). Their analyses of morphology, allozymes, and DNA sequences resulted in basically two different topologies of species relationships – one topology supported by morphological characters, many of which were unusual relative to other salamanders due to cave adaptations, and another general topology with different sisterspecies relationships based on allozymes and DNA sequences. The questions were how does one separate whether or not one of the two trees is correct, is one more likely due to convergence than to common ancestry, or are both topologies incorrect (another possibility)? To aid their own research and that of others, Wiens et al. (2003) advanced a series of tests to help distinguish between convergence and homology. These include (1) high degree of support for the convergent clade, (2) an association between the putative convergent characters and the ecological setting of species possessing these characters, (3) phylogenetic evidence that the convergent clade is wrong. With respect to the first criterion, Britz and Conway (2009) provide a series of morphological characters that they hypothesize are synapomorphic for at least Danionella, Sundadanio, and Paedocypris, that in the current analysis based on nuclear genes are interpreted as independently evolved character losses or changes. As for the other morphological characters identified by Britz and Conway (2009), these can be explained by a lack of a broader perspective in taxon sampling to accurately place characters in their correct hierarchical position in the evolution of Paedocypris and remaining Cypriniformes. Criterion 2 seeks an association between the putative phenotype and some ecological or environmental condition. For the miniature Cypriniformes, the only common environmental characteristic might be their association to varying degrees of tannin-stained acidic waters with limited current. However, this is strictly speculative and may have no impact on the developmental biology of these species or the maintenance of apomorphic losses or changes of characters. This is an extremely important area of investigation and could easily be examined, especially with studies involving the model species Danio rerio. Given the relatively ease of care of the miniature species in captivity, this is one series of ecolog-

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ical parameters that could be repeatedly tested, unless its evolution occurred too far in the past and the development is canalized. Under Criterion 3, we have clearly demonstrated using multiple nuclear genes that miniaturization in Cypriniformes has occurred multiple times in different and unrelated clades. Further, more dense taxon sampling within the Rasborine Clade and other areas of the Cypriniformes Tree of Life where miniature species occur will be necessary to elucidate in more detail any ancestral-state reconstructions of the various anatomical characters associated with the various origins of miniaturization to seek common explanations for this phenomenon. Thus, the situation of miniaturization in cypriniform fishes appears to be another example of convergent evolution of similar phenotypes to environmental selection as described by Wiens et al. (2003) for cave-adapted salamanders. It is suspected that the systematic and evolutionary biology communities will benefit greatly from more detailed and common studies of these two groups of vertebrates and research linkages to the developmental biology and genomics communities currently involved with anatomical ontologies of both amphibians and fishes.

4.4. Closing thoughts At least two additional observations are worthy of discussion regarding the miniaturization of species in the Cypriniformes. That is, future research should clearly delineating the quality of miniature in a species and link studies of the independent evolution of miniaturization in Cypriniformes to ongoing genetic and morphological studies with Danio rerio. As clearly outlined by McClain and Boyer (2009) the evolution of body size can occur through a variety of mechanisms, some of which may be underlying the evolution of miniature cypriniforms. Considering all miniature Cypriniformes to possess the same quality ‘‘miniature” as is done by using the definition or criterion of Weitzman and Vari (1988) (species reproducing 620 mm, or if reproductive information are not available, species less than 26 mm) is equivalent to approaching a question in need of a microscope with a hand lens. Further elucidating the evolution of a miniature state or quality requires more detailed comparisons of the species in question relative to their sister taxa to see if the quality has been acquired in the same manner. The explanatory powers of genomics linked to morphologies are only beginning to be realized in fishes, now through the development of anatomical ontologies associated with Danio rerio, Cypriniformes fishes through the Cypriniformes Tree of Life (Mabee et al., 2007a,b; Mayden et al., 2007) and Phenoscape initiative (www.phenoscape.org; Dahdul et al., 2010a,b; Balhoff et al., 2010). The potential for advancement of our understanding of various aspects of evolutionary biology, especially the underlying genetic mechanisms of morphological change in evolution, are seemingly unlimited through the adoption of collaborative studies involving genomics, continued support of detailed morphological studies, and a clear investment in the development of various ontologies. With bioinformatics, genomics, and efforts in developing mutant phenotypes derived from studies from the model species Danio rerio that are linked to wild-type phenotypes observed in systematic studies via ontologies and the zebrafish databases, the evolutionary, genetic, and morphological communities can all benefit in their search for answers to common questions (see Section 4.2 and Fig. 6). Additionally, what is needed to further advance this field is a closer association between the molecular and morphological systematists, a resurgence in interest in anatomical research and detailed anatomical studies, and increased communication and collaboration between the systematic and evolutionary biology communities with researchers studying model organisms and the

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development and emergence of varied ontologies of these organisms. 5. Paedocyprididae, new family; Paedocypridoidea, new superfamily 5.1. Diagnosis The diagnostic characters used for the type genus Paedocypris Kottelat et al., (2006: 895–896) are considered valid for diagnosing this family. ‘‘The following characters of Paedocypris are unique among fishes: outermost pelvic-fin ray of male highly modified with ventral hemitrich greatly expanded, flattened, supporting keratinized skin-pad and tip of dorsal hemitrich supporting small hook-like projection of keratinized skin directed outwards (Fig. 3a); abductor and ventral arrector muscles of pelvic girdle of male hypertrophied (Fig. 3b), the former attached to ventral extremity of os suspensorium (versus not hypertrophied and abductor muscle restricted to basipterygia); pad of keratinized skin in front of pelvic fin in male (Figs. 2a, c and 3a, c). The following characters are unique within Cypriniformes: presence in adults (versus only in larvae) of a long post-anal larval-fin-fold along ventral edge of caudal peduncle, from posterior extremity of anal-fin base to caudal-fin base (Fig. 2); basipterygium of male hypertrophied (versus not hypertrophied) (Fig. 4); abdominal vertebrae 7– 13 with short haemal spines (versus haemal spines only on caudal vertebrae); genital papilla of male hypertrophied, in form of small bag surrounding and including anterior 2 or 3 anal-fin rays, posteriorly confluent with fin membrane (Fig. 2c); pharyngeal teeth tricuspid. The following characters are also diagnostic, although not unique to the genus: miniature adult size (maximum 10.5 mm); males with large conical tubercles along dentary, preopercle/interopercle, subopercle/opercle, frontal and lachrymal; caudal peduncle very slender; dorsal-fin rays ii,3,ii or ii,4,i; anal-fin rays iii,5,ii, iii,6,i, iii,5,iii, iii,6,ii, or iii,7,i; caudal-fin slightly emarginate, with 14 branched rays; 5 pelvic-fin rays; 8 pectoral-fin rays; 12K13 abdominalC21K23 caudalZ33K35 vertebrae; absence of the following bones and cartilages in the neurocranium (parietals, nasals, vomer, preethmoid, circumorbitals (except lachrymal)), in the lower jaw (angular), in the upper jaw (bony kinethmoid), in the hyopalatine arch (ectopterygoid), in the shoulder girdle (post-temporal, post-cleithrum, cartilaginous distal pectoral radials) and in the axial skeleton (supraneural 2, epural); frontals narrow leaving the brain exposed; intercalarium reduced to a splint; basibranchials, ceratobranchials, epibranchials and pharyngobranchials not ossified, except ceratobranchials 4 and 5; proximal–middle radials of dorsal and anal-fins remain mainly cartilaginous; scales absent. Coloration in life: translucent orange; males with orange iridescent spot between eyes and one on nape; preserved, yellowish grey with black pigments organized as in Figs. 1 and 2.” 5.2. Composition This family currently includes three species of Paedocypris, P. progenetica, and P. micromegethes, both described by Kottelat et al. (2006) and P. carbunculus described by Britz and Kottelat (2008). Other undescribed species are known to exist and await formal description. Future inventory efforts from southeast Asia will likely reveal many additional species in this clade because of the understudied habitats that they are thought to be restricted. Future phylogenetic efforts may also identify additional taxa never before examined in a phylogenetic context to be part of this family and potentially providing valuable insight in comparative evolutionary studies as to the mechanisms involved in the origin and maintenance of their miniaturized state.

5.3. Distribution Currently, this family is only reported in Southeast Asia from Indonesia and Malaysia. However, given the habitat profile for species of Paedocypris and the fact that these areas have been poorly inventoried, future inventory efforts will likely find additional, related taxa in other surrounding geographic areas. 6. Sundadanionidae, new family 6.1. Diagnosis Diagnostic morphological characters for the family derived, in part, from Kottelat and Witte (1999) and Britz and Conway (2009). A clade of cypriniformes with a maximum size reported to be 22.5 thick mm SL; diagnosed by the presence of a semicircular indentation on the ventromedian flange of the dentary; five branched anal rays; fourth centrum of the Weberian apparatus extending well below the fused second and third centra; conspicuous sexual dichromatism; ability to generate repeated croaking sounds when disturbed; barbels and lateral line pores absent; lateral line pores absent; barbels absent; males with large posterior flange on cleithrum, serrate dorsal flange on pectoral fin ray 5; co-ossified scapulo-coracoid, hypertrophied outer arm of os suspensorium, enlarged rib 5, and enlarged muscle between os suspensorium and rib 5. 6.2. Composition The family currently contains only one species, Sundadanio axelrodi (Brittan, 1976); however, given the distribution of this species and the paucity of comprehensive sampling of these regions it is likely that new species will be discovered or S. axelrodi will likely be found to be a composite of multiple species. 6.3. Distribution Endemic to Borneo (Sarawak, Kalimantan Barat, Kalimantan Tengah), Bangka and Sumatra (Jambi, Riau). 7. Tanichthyidae, new family 7.1. Diagnosis A clade of cypriniformes with laterosensory canals on head and lateral line absent; barbels, symphyseal knob on lower jaw, and ventral abdominal keel absent; pharyngeal teeth in two rows; anterior and posterior narial openings confluent, forming a single, elongate opening; dorsal fin base short, fin without spine-like first ray; anal fin base short and not bordered by enlarged row of scales; body moderately compressed; mouth oblique, lower jaw projecting slightly beyond upper jaw; centra of second and third vertebrae not fused to one another; breeding adult males with four to eight cornified tubercles in a single tooth-like row. 7.2. Composition The family currently contains three species, including the popular aquarium species White Cloud Mountain Minnow, Tanichthys albonubes Lin, 1932 (type species), T. micagemmae Freyhof and Herder (2001), and T. thacbaensis Nguyen and Ngo (2001). 7.3. Distribution China and Vietnam.

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literature for various possible family group names and his help in formulating these names. Dr. Kobayashi provided specimens of Paedocypris and photographs. Dr. Zeb Hogan kindly provided the color photograph of Catlocarpio siamensis. This research was supported by the USA National Science Foundation grant to R.L. Mayden (EF-0431326).

Acknowledgments We thank Gloria Arratia and Kevin Conway for discussions of morphological characters. Kevin Conway read an earlier draft of this paper and but does not agree with our conclusions. We also thank Kevin Tang for discussions on the historical taxonomic Appendix A

Taxa included in this study and accession numbers of sequences in GenBank. Family

Taxon

GenBank accession number RAG1

RH

IRBP

EGR1

EGR2B

EGR3

EU409728 FJ650438 EU409729 FJ650439

EU409760 FJ650454 EU409761 FJ650455

Outgroups Gonorynchidae Bagridae Characidae Alestiidae

Gonorynchus greyi Pseudobagrus tokiensis Chalceus macrolepidotus Phenacogrammus interruptus

EU409606 FJ650410 EU409607 FJ197124

EU409632 FJ197075 EU409633 FJ197073

EU409664 EU409665 FJ197123

EU409696 FJ650422 EU409697 FJ650423

Cypriniformes Cobitoidea Balitoridae Balitoridae Catostomidae Catostomidae Botiidae Botiidae Botiidae Botiidae Cobitidae Cobitidae Cobitidae Cobitidae Cobitidae Gyrinocheilidae Gyrinocheilidae Nemacheilidae Nemacheilidae Nemacheilidae Nemacheilidae Nemacheilidae Nemacheilidae Nemacheilidae Vaillantellidae

Sewellia lineolata Homaloptera parclitella Catostomus commersoni Cycleptus elongatus Botia dario Leptobotia pellegrini Syncrossus beauforti Yasuhikotakia morleti Acantopsis sp. Cobitis takatsuensis Niwaella multifasciata Pangio oblonga Somileptus gongota Gyrinocheilus aymonieri Gyrinocheilus pennocki Acanthocobitis sp. Barbatula barbatula Lefua costata Oreonectes platycephalus Schistura savona Traccatichthys pulcher Triplophysa gundriseri Vaillantella maassi

EU409609 EU409610 EU409612 EU409613 EU409614 EU292683 FJ650411 FJ650412 FJ650413 EU409616 EU409615 EU711141 FJ650414 EU292682 FJ650415 FJ650416 EU711107 EU409608 FJ650418 FJ650419 EU409611 FJ650420 EU711132

EU409635 EU409636 EU409638 EU409639 EU409641 EU409640 FJ650470 FJ650471 FJ650472 EU409643 EU409642 FJ197041 FJ650473 FJ197071 FJ650474 FJ650475 FJ650476 EU409634 FJ650478 FJ650479 EU409637 FJ650480 FJ197031

EU409667 EU409668 EU409670 EU409671 EU409673 EU409672 FJ650482 FJ650483 FJ650484 EU409675 EU409674 FJ197091 FJ650485 FJ197122 FJ650486 FJ650487 FJ650488 EU409666 FJ650490 FJ650491 EU409669 FJ650492 FJ197080

EU409699 EU409700 EU409702 EU409703 EU409705 EU409704 FJ650424 FJ650425 FJ650426 EU409707 EU409706 FJ650427 FJ650428 EU409727 FJ650429 FJ650430 FJ650431 EU409698 FJ650433 FJ650434 EU409701 FJ650435 FJ650437

EU409731 EU409732 EU409734 EU409735 EU409737 EU409736 FJ650440 FJ650441 FJ650442 EU409739 EU409738 FJ650443 FJ650444 EU409759 FJ650445 FJ650446 FJ650447 EU409730 FJ650449 FJ650450 EU409733 FJ650451 FJ650453

EU409763 EU409764 EU409766 EU409767 EU409769 EU409768 FJ650456 FJ650457 FJ650458 EU409771 EU409770 FJ650459 FJ650460 EU409791 FJ650461 FJ650462 FJ650463 EU409762 FJ650465 FJ650466 EU409765 FJ650467 FJ650469

Acheilognathus tabira Paracheilognathus himantegus Rhodeus ocellatus kurumeus Aphyocypris chinensis Ischikauia steenackeri Megalobrama amblycephala Macrochirichthys macrochirus Opsariichthys uncirostris Paralaubuca typus Yaoshanicus arcus Zacco sieboldii Acrossocheilus paradoxus Barbonymus gonionotus Barbus callipterus Garra spilota Gymnocypris przewalskii

EU409617 EU409618

EU409644 EU409645

EU409676 EU409677

EU409708 EU409709

EU409740 EU409741

EU409772 EU409773

EU711142 EU292692 EU292687 EU409620 EU409630

FJ197043 FJ197066 EU409648 EU409647 EU409659

FJ197093 FJ197117 EU409680 EU409679 EU409691

FJ531277 FJ531256 EU409712 EU409711 EU409723

FJ531306 FJ531285 EU409744 EU409743 EU409755

FJ531335 FJ531314 EU409776 EU409775 EU409787

FJ197126 EU409619 FJ531254 EU292713 FJ531245 FJ531246 FJ531247 EU409621 EU711149

FJ197068 EU409646 FJ531361 FJ197069 FJ531342 FJ531344 FJ531345 EU409649 FJ197051

FJ197119 EU409678 FJ531380 FJ197120 FJ531362 FJ531364 FJ531365 EU409681 FJ197102

FJ531271 EU409710 FJ531282 FJ531283 FJ531255 FJ531258 FJ531259 EU409713 FJ531265

FJ531300 EU409742 FJ531311 FJ531312 FJ531284 FJ531287 FJ531288 EU409745 FJ531294

FJ531329 EU409774 FJ531340 FJ531341 FJ531313 FJ531316 FJ531317 EU409777 FJ531323

Cyprinoidea Acheilognathidae Acheilognathidae Acheilognathidae Cultrine clade Cultrine clade Cultrine clade Cultrine clade Cultrine clade Cultrine clade Cultrine clade Cultrine clade Cyprinidae Cyprinidae Cyprinidae Cyprinidae Cyprinidae

(continued on next page)

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Appendix A (continued) Family

Cyprinidae Cyprinidae Cyprinidae Gobionidae Gobionidae Gobionidae Gobionidae Gobionidae Gobionidae Leptobarbidae Leuciscidae Leuciscidae Leuciscidae

Taxon

GenBank accession number RAG1

RH

IRBP

EGR1

EGR2B

EGR3

EU409623 EU409622 EU292685 EU409626 EU292689 EU711154 EU409624 EU409625 FJ531252 FJ531249 EU292691 EU711144 EU409627

EU409651 EU409650 FJ531356 EU409654 FJ197056 FJ197057 EU409652 EU409653 FJ531358 FJ531351 EU409657 FJ197045 EU409655

EU409683 EU409682 FJ531375 EU409686 FJ197107 FJ197108 EU409684 EU409685 FJ531377 FJ531371 EU409689 FJ197095 EU409687

EU409715 EU409714 FJ531275 EU409718 FJ531264 FJ531266 EU409716 EU409717 FJ531278 FJ531268 EU409721 FJ531272 EU409719

EU409747 EU409746 FJ531304 EU409750 FJ531293 FJ531295 EU409748 EU409749 FJ531307 FJ531297 EU409753 FJ531301 EU409751

EU409779 EU409778 FJ531333 EU409782 FJ531322 FJ531324 EU409780 EU409781 FJ531336 FJ531326 EU409785 FJ531330 EU409783

Leuciscidae Leuciscidae Paedocyprididae Paedocyprididae Psilorhynchidae Psilorhynchidae Rasborine clade Rasborine clade Rasborine clade Rasborine clade Rasborine clade Rasborine clade Rasborine clade Rasborine clade Rasborine clade

Hampala macrolepidota Labeo chrysophekadion Puntius titteya Biwia zezera Gobio gobio Hemibarbus barbus Romanogobio ciscaucasicus Sarcocheilichthys parvus Squalidus chankaensis Leptobarbus hoevenii Notropis baileyi Pelecus cultratus Phoxinus perenurus sachalinensis Scardinius erythrophthalmus Semotilus atromaculatus Paedocypris sp. 1 Paedocypris sp. 2 Psilorhynchus sucatio Psilorhynchus homaloptera Aspidoparia morar Barilius bendelisis Boraras merah Chela dadiburjori Danio erythromicron Danio margaritatus Danio albolineatus Danio dangila Danio rerio

EU409628 EU409629 GQ365218 GQ365219 FJ531251 FJ531250 EU711105 EU292693 EF452838 EU292694 EU292698 EU292695 EU292696 EU292697 U71093

EU409656 EU409658 GQ365226 GQ365227 FJ531355 FJ531354 FJ531343 FJ531346 GQ365220 GQ365221 GQ365222 GQ365223 EU409661 EU409660 L11014

EU409688 EU409690 GQ365262 GQ365263 FJ531374

EU409752 EU409754 GQ365244 GQ365245 FJ531303 FJ531302 FJ531286 FJ531289 GQ365238 GQ365239 GQ365240 GQ365241 EU409757 EU409756 NM_130997

EU409784 EU409786 GQ365253 GQ365254 FJ531332 FJ531331 FJ531315 FJ531318 GQ365247 GQ365248 GQ365249 GQ365250 EU409789 EU409788 scaffold2320.1

Rasborine clade Rasborine clade Rasborine clade Rasborine clade Rasborine clade Rasborine clade Rasborine clade Rasborine clade Rasborine clade Rasborine clade Rasborine clade Sundadanionidae Tanichthyidae Tincidae

Danionella sp. Devario regina Esomus longimanus Horadandia atukorali Luciosoma setigerum Microrasbora kubotai Microrasbora nana Microrasbora rubescens Rasbora bankanensis Rasbora steineri Trigonostigma heteromorpha Sundadanio axelrodi Tanichthys albonubes Tinca tinca

EU292700 EU292701 FJ531248 EU292703 EU292704 EU292707 EU292705 EU292706 EU292709 EU409631 EU292712 EU292711 FJ531253 EU711162

FJ531347 FJ531348 FJ531349 FJ531350 FJ531352 FJ531353 GQ365224 GQ365225 FJ531357 EU409662 FJ531360 GQ365228 FJ531359 FJ197070

FJ531367 FJ531368 FJ531369 FJ531370 FJ531372 FJ531373 GQ365260 GQ365261 FJ531376 EU409694 FJ531379 GQ365264 FJ531378 FJ197121

EU409720 EU409722 GQ365235 GQ365236 FJ531274 FJ531273 FJ531257 FJ531260 GQ365229 GQ365230 GQ365231 GQ365232 EU409725 EU409724 NM 131248 FJ531261 FJ531262 FJ531263 FJ531267 FJ531269 FJ531270 GQ365233 GQ365234 FJ531276 EU409726 FJ531281 GQ365237 FJ531279 FJ531280

FJ531290 FJ531291 FJ531292 FJ531296 FJ531298 FJ531299 GQ365242 GQ365243 FJ531305 EU409758 FJ531310 GQ365246 FJ531308 FJ531309

FJ531319 FJ531320 FJ531321 FJ531325 FJ531327 FJ531328 GQ365251 GQ365252 FJ531334 EU409790 FJ531339 GQ365255 FJ531337 FJ531338

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FJ531363 FJ531366 GQ365256 GQ365257 GQ365258 GQ365259 EU409693 EU409692 X85957

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