Geographic distributions, phenotypes, and phylogenetic relationships of Phalloceros (Cyprinodontiformes: Poeciliidae): Insights about diversification among sympatric species pools

Accepted Manuscript Geographic distributions, phenotypes, and phylogenetic relationships of Phalloceros (Cyprinodontiformes: Poeciliidae): insights about diversification among sympatric species pools Andréa T. Thomaz, Tiago P. Carvalho, Luiz R. Malabarba, L. Lacey Knowles PII: DOI: Reference:

S1055-7903(18)30527-X https://doi.org/10.1016/j.ympev.2018.12.008 YMPEV 6364

To appear in:

Molecular Phylogenetics and Evolution

Received Date: Revised Date: Accepted Date:

13 August 2018 6 December 2018 6 December 2018

Please cite this article as: Thomaz, A.T., Carvalho, T.P., Malabarba, L.R., Lacey Knowles, L., Geographic distributions, phenotypes, and phylogenetic relationships of Phalloceros (Cyprinodontiformes: Poeciliidae): insights about diversification among sympatric species pools, Molecular Phylogenetics and Evolution (2018), doi: https://doi.org/10.1016/j.ympev.2018.12.008

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Title: Geographic distributions, phenotypes, and phylogenetic relationships of Phalloceros (Cyprinodontiformes: Poeciliidae): insights about diversification among sympatric species pools

Authors: Andréa T. Thomaz1, Tiago P. Carvalho 2,3, Luiz R. Malabarba2, L. Lacey Knowles1 1

Department of Ecology and Evolutionary Biology, University of Michigan, 1105 N. University,

48109, Ann Arbor MI, USA. 2

Laboratório de Ictiologia, Departamento de Zoologia, Universidade Federal do Rio Grande do

Sul. Av. Bento Gonçalves, 9500, 91501-970 Porto Alegre RS, Brazil. 3

Research Associate, Department of Ichthyology, The Academy of Natural Sciences of

Philadelphia, Drexel University, 1900 Benjamin Franklin Parkway, 19103 Philadelphia, PA, USA.

Corresponding Author and current address: Andréa T. Thomaz, [email protected], 778-316-9657 Biodiversity Research Centre and Department of Zoology University of British Columbia 2212 Main Mall Vancouver, BC – Canada, V6T 1Z4

Running Title: Phylogeny and diversification of Phalloceros

Declarations of interest: none Word count: 6,019

1

ABSTRACT With 22 described species, Phalloceros is the most species-rich genus of Poeciliidae in South America. Phalloceros diversity is characterized by high degrees of endemism and sympatry in coastal and inland drainages in southeastern South America. The taxa are also characterized by pronounced differentiation in sexual characters (i.e., female urogenital papilla and male gonopodium), which might have contributed to their diversification. Here we estimate phylogenetic relationships based on more than 18,000 loci in 93 individuals representing 19 described species and two putative undescribed species. Morphologically defined species correspond to monophyletic species lineages, with individuals within a species clustering together in phylogenetic estimates, with the main exception being P. harpagos, supporting undiscovered diversity in this morphospecies. Shifts in the female and male sexual traits (i.e., urogenital papilla and gonopodium) occurred in concert multiple times along the phylogeny highlighting the role of sexual selection in driving divergence in this genus. Out of 22 valid species, 14 species are found in sympatry with at least one other species of this genus. However, most co-occurrences are observed among non-sister species suggesting that diversification among closely related species involved mostly allopatric speciation, with only two instances of sympatric sister-species observed. A strong mismatch in sexual traits among sympatric taxa suggests that co-existence may be linked to divergent sexual traits that maintain species genetic distinctiveness through mechanical disruptions of interbreeding.

KEY-WORDS: ddRADseq, dusky millions poeciliids, female urogenital papilla, male gonopodium, sexual selection, South America.

2

1. INTRODUCTION Species rich clades are often targets of phylogenetic analysis because these frameworks are critical to understanding their history of diversification. For example, understanding lineages relationships was essential for inferring the rapid radiation of African cichlids (Wagner et al., 2013), ecological speciation of stick-insects (Nosil et al., 2012) and the role of dispersal and vicariant events in the radiation of placental mammals (Murphy et al., 2001). Yet for the Neotropical ichthyofauna, the most diverse vertebrate fauna on the planet (~6,000 sp.; Reis et al., 2016), significant gaps in our knowledge of species-level phylogenetic relationships are an impediment to understanding the mechanisms of diversification contributing to this rich fauna (Vari and Malabarba, 1998; Albert et al., 2011). Among the species-rich Neotropical freshwater fish fauna, the poeciliids stand out. It is a relatively species rich group (274 valid species; Fricke et al., 2018), but they are also known as examples of rapid ecological divergence and sexual selection in the speciation process (e.g., Gambusia, Poecilia and Xiphophorus; Endler et al., 1983; Crispo et al., 2006; Pollux et al., 2014). Much of this attention is on their interesting reproductive life history as live bearing fish with internal fertilization (Langerhans et al., 2005; Pollux et al., 2009). However, such studies have mostly focused on poeciliids in Central America and West Indies islands. South American poeciliids (Lucinda, 2005a, 2005b, 2008; Lucinda et al., 2005), which include those in the early diversification of this group (Hrbek et al., 2007), have received relatively little attention, and lack molecular phylogenetic studies. Our work addresses this knowledge gap, presenting a molecular analysis of Phalloceros, a diverse genus with co-distributed species and pronounced differentiation in sexual characters.

3

The South American genus Phalloceros, popularly known as the dusky millions poeciliids, is a species-rich genus whose diversity remained hidden for more than a century (Lucinda, 2008) under the cover of a single described species, Phalloceros caudimaculatus (Hensel, 1868; Eigenmann, 1907). Cladistic analysis of morphological data (Lucinda and Reis, 2005; Lucinda, 2008) demonstrated the monophyly of the genus, proposed intrageneric relationships (Fig. 1), and was the basis for 21 newly described species that were diagnosed (mostly) by sexual traits (e.g. male gonopodium – the inseminating organ, and female urogenital papilla), color pattern and fin-ray counts (Table 1; Fig. 1). With 22 described species, Phalloceros is now recognized as the most species-rich South American genus of Poeciliidae, and it’s diversity ranks fourth among all poeciliid genera, behind Gambusia (42 spp.; Langerhans et al., 2012), Poecilia (40 spp.; Froese and Pauly, 2017) and Xiphophorus (24 spp.; Kalmann et al., 2004). There is also the potential of yet to be discovered diversity, such as the intraspecific morphological variability in the broadly distributed species Phalloceros harpagos (see Lucinda, 2008). Likewise, with only a few strictly allopatric species, phylogenetic study is needed to develop a framework for inferences about the speciation process. For example, Phalloceros species are characterized by high degrees of endemism and sympatry (Lucinda, 2008), with most taxa confined to few riverine systems (e.g., rivers draining to Paranaguá and Babitonga bays) of coastal and continental drainages in the southeastern portion of South America (Fig. 2). This distribution has led to hypotheses that focus on a mechanism of diversification linked to repeated events of faunal exchange across basins, as illustrated by freshwater taxa from this area (Weitzman and Weitzman, 1988; Ribeiro, 2006; Ribeiro et al., 2006, Thomaz et al., 2015, 2017; Lima et al., 2017). However, with many Phalloceros species sympatrically distributed and often differentiated in their sexual traits (Table 1; Lucinda, 2008),

4

evolution of genitalia, perhaps in concert with ecologically driven divergence (e.g., Jennions and Kelly, 2002; Oneal and Knowles, 2013), and/or co-evolution between male and female (e.g., lock-and-key mechanism, or antagonistic selection and female preference; Anderson and Langerhans, 2015), may contribute to their diversification. Here we estimate phylogenetic relationships based on genomic data and use this framework to examine the extent to which there is concerted evolution of male and female genitalia, and how sympatric species differ in these traits. In addition, with multiple individuals sequenced per taxon, we also assess whether species form monophyletic lineages, and use the distribution of species to draw insights about the geography of Phalloceros diversification.

2. MATERIAL AND METHODS 2.1. Taxon sampling We sampled 96 individuals representing 19 of the 22 putative species of Phalloceros (Lucinda, 2008), plus two putative, undescribed species (see below). Tissue vouchers and accompanying specimens are housed at the following institutions: Museu de Ciências e Tecnologia, Pontifícia Universidade Católica do Rio Grande do Sul (MCP), Porto Alegre Brazil; and Departamento de Zoologia, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre - Brazil (Supplementary Table S1). Specimens were loaned to ATT at University of Michigan following Brazilian legislation under Material Transfer Agreement (MTA 02/2011, 01/2013 and 03/2013). A minimum of two and a maximum of nine individuals per species sampled across their respective distribution ranges were included in phylogenetic analyses. Nine of the 96 samples studied were paratypes of five species (for detailed information about samples see Supplementary Table S1).

5

All specimens were identified following the taxonomic key and external morphology diagnosis proposed by Lucinda (2008). In some cases, the identification could not be confirmed given a mismatch between species distribution and sample localities, and putative identifications of morphotypes that did not have the full complement of characters for a particular species diagnosis. There were four such cases, including (i) P. cf. mikrommatos that was identified based on its geographic distribution since it is the only species occurring at João de Tiba River drainage, Bahia State, Brazil, but couldn’t be confirmed based on a mismatch in its diagnostic number of anal-fin rays in females; (ii) P. cf. uai, which lacked the unique squared-shape lateral spot on the side of the body, despite having the remaining diagnostic characters of this species, and is the only species of the genus distributed in the São Francisco River basin, Minas Gerais State, Brazil; (iii) a paratype specimen of P. buckupi that was re-identified as P. titthos based on the combination of a straight female urogenital papilla and small papillae in the lower lip (we noticed that this lot of P. buckupi paratypes, MCP 31190, is mixed, and contained individuals of both species); and (iv) two putatively distinct species from coastal streams near Ubatuba, São Paulo State that do not fit the diagnostic traits of any described species, which we refer to as “P. sp. R” and “P. sp. L”, based on a right and left direction of female urogenital papilla, respectively.

2.2. RADseq genomic data generation and processing Genomic DNA was extracted from tissue samples using DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) following manufacturer’s protocol for 96 samples. A doubledigest reduced representation library (ddRAD) was constructed (for details, see Peterson et al., 2012) with 500 ng of genomic DNA from each individual. Briefly, genomic DNA was double

6

digested with EcoRI and MseI restriction enzymes, followed by a ligation step to incorporate unique barcodes (10 bp). Ligation products were pooled together and fragments between 300400 bp were size-selected with Pippin Prep (Sage Science, Beverly, MA, USA). The fragments were amplified by eight cycles of PCR to incorporate the Illumina flowcell adaptor. Each step was followed by a clean-up with AMPure beads (using a 1.6x beads/DNA ratio; except after size selection) to remove small DNA fragments and a quantification assay using Qubit. The library was sequenced at the Centre for Applied Genomics (Hospital for Sick Children, Toronto, ON, Canada) in one lane of Illumina HiSeq2500 platform for 150 bp single-end reads. The program ipyrad v. 0.6.17 (Eaton, 2014; https://github.com/dereneaton/ipyrad/blob/master/docs/index.rst) was used to demultiplex and process the genomic reads. Briefly, demultiplexed (step 1) data files were identified by their barcodes, allowing for a maximum of two mismatches per barcode. Reads were filtered (step 2) by converting base calls with quality score <20 to Ns and discarding the ones with >5 Ns, Illumina adapters were excluded, and sequences trimmed to a minimum length of 110 bp. This was followed by the mapping of reads within-samples and alignments (step 3) using the de novo and + reference genome in ipyrad. For the reference genome, ipyrad uses bwa (Li and Durbin, 2009) under the default parameters of ‘bwa mem’ option. Xiphophorus maculatus was used as the reference genome [GenBank accession no: AGAJ00000000.1 (Schartl et al., 2013)] and sequence similarity of 0.9 was used to identify reads as homologous and cluster them. Afterwards, heterozygosity and sequencing error rate were estimated (step 4) based on a maximum of two alleles (diploid) per cluster. These estimates were applied under a binomial model to assemble and filter the consensus sequences within each cluster (step 5), with a minimum depth of six and a maximum of five ambiguous (Ns) bases in each consensus

7

sequence. These consensus sequences were then clustered across samples (step 6) with the same thresholds applied for within-samples cluster (step 3; see above). In the final step of ipyrad (step 7), two datasets were created: the full dataset that included all samples of Phalloceros and a subsample dataset, with one individual per species. For the full dataset (93 samples), a final filter was performed per locus based on a minimum of 10 samples, maximum of eight indels, maximum of 20 SNPs per loci, and a maximum 0.5 proportion of shared heterozygotes sites. For the subsample dataset, the only difference was that we selected one individual per species with the larger number of reads (21 samples in total). Detailed per sample information about each ipyrad step is available in Supplementary Table S2. Shared loci among taxa were visualized with the corrplot package in R (R Core Team, 2017; Wei et al., 2017).

2.3. Phylogenetic inference and evolutionary history of sexual characters Evolutionary relationships were estimated using the full dataset under two approaches. A maximum likelihood (ML) estimate was obtained from RAxML v. 8.2.8 (Stamatakis, 2014) under a GTR+CAT nucleotide substitution model for a concatenated dataset of all loci. A coalescent-based approach that accounts for differences in the genealogical history of loci was also used to estimate phylogenetic relationships. For this analysis, unlinked SNPs (i.e., a single SNP per locus) were analyzed using the program SVDquartets (Chifman and Kubatko, 2014) as implemented in PAUP* 4.0 (Swofford, 2003). Two phylogenetic trees were estimated with SVDquartets: one for the phylogenetic relationships estimated among individuals, and another for species lineages with multiple individuals sampled from morphologically delimited species (or as in the case of P. harpagos, geographically separate populations). In both cases exhaustive

8

quartet sampling was performed (2,919,734 quartets) and support was assessed with 1,000 bootstrap replicates in all reconstructions. To infer the position of the root in Phalloceros, we extracted the SNPs from the aligned reference genome that corresponded to those in our sequenced individuals from the subsample dataset. We then reconstructed the phylogenetic relationships among individuals using unlinked SNPs with SVDquartets setting Xiphophorus maculatus as outgroup. The result of this analysis was used to set the root position in analyses using the full dataset (see above). To explore how sexual characters and color pattern evolved, we performed ancestral state reconstructions of the female urogenital papilla (i.e., straight along median ventral line, turned to the right or turned to the left - character 142 in Lucinda, 2008; Fig. 3), male gonopodium (i.e., no hooks, one hook on the left half of the paired appendix, or hooks on both halves, either positioned outward or inward on the paired appendix of the gonopodium – characters 95 and 98 in Lucinda, 2008; Fig. 3), and the presence and shape of the lateral spot(s) (i.e., rounded, elliptical, elliptical showing an additional spot on caudal peduncle, vertically elongated, and absent - character 134 in Lucinda, 2008; Fig. 1). For these analyses, ancestral character state estimates were performed under three separate models of discrete character transitions (ERequal rates, SYM – symmetrical and ARD – all rates different) using the ape package in R (Paradis et al., 2004). The concatenated tree inferred with RAxML was pruned to a single terminal per species and transformed to an ultrametric tree with the chronopl tool in ape using a crosschecked smoothing parameter (λ = 1) based on penalized likelihood (Sanderson, 2002). To identify the best model for each reconstruction, transition models with different number of parameters were compared with a Chi-square test. In all cases the selected model was the one with equal rate transitions (ER; Table S3) among character states.

9

All genomic library processing and phylogenetic inferences were run under parallel execution through computational resources and services provided by Advanced Research Computing at the University of Michigan flux facility, Ann Arbor, MI, USA. Input files (e.g., nexus and phylip files) and phylogenetic reconstructions are available at the University of Michigan DeepBlue repository (http://dx.doi.org/10.7302/Z2G44NJ2; Thomaz et al., 2018 [DATA SET]).

2.4. Geography of species divergence To evaluate putative cases of allopatric speciation between sister species versus potential sympatric speciation within a river basin we mapped species distributions based on data from Lucinda (2008) and our additional records (see Supplementary Table S4). We also note when codistributed taxa from the same river basin do not co-occur (e.g. they occupy the lower vs. upper segments of the river; Table 1).

3. RESULTS 3.1. ddRAD data summary A total of 91,593,452 raw reads were generated, of which 86,077,586 with matching barcodes were retained. An average of 896,642 (±438,299) reads were generated per sample, except for three of the 96 samples with < 100,000 reads, which were removed; all analyses were performed with 93 individuals for 19 species plus two putative new species. After data processing in ipyrad, we obtained a dataset with 18,122 loci (a total of 2,543,931 bp) and an average of 7,880 (±1122) loci per individual (see Supporting Table S2 for information per

10

individual). A final dataset with 58% missing data was used for the phylogenetic reconstructions, with loci homogeneously shared between samples (Fig. S1).

3.2. Phylogenetic relationships All phylogenetic estimates recovered three large clades, which we refer to as clades A, B and C (Figs. 3, S2, S3, S4 and S5). The one exception is P. enneaktinos, which is a member of clade A based on the species-tree analysis and concatenated dataset (Figs. 3 and S2), but is in clade C in the coalescent-based analysis of individuals (Fig. S3). Within each clade, topological agreement and high support values of relationships are estimated towards the tree tips across methods, whereas deeper relationships within the A, B, C clades differ between the concatenated dataset and the coalescent-based estimates (Fig. 3 and red nodes in Figs. S2, S3 and S4). Individuals within species form monophyletic lineages across analyses, except for Phalloceros elachistos, which is paraphyletic in both analyses at the individual level based on the concatenated dataset and under the coalescent, and P. harpagos, whose individuals are scattered across the tree. Otherwise, not only did individuals tend to be most closely related to conspecifics diagnosed by morphology, but such relationships were also supported by high bootstrap values (Figs. S2 and S3).

3.3. Phylogenetic distribution of changes in morphological characters The position of the female urogenital papilla is observed as three discrete states: straight, right turned, and left turned (Fig. 3). While a single shift to left-turned papilla is observed (Phalloceros sp. L, P. aspilos, and P. tupinamba), reconstructions suggests three probable shifts between straight and right turned urogenital papilla in females of B and C clades with a straight

11

directed papillae inferred as the ancestral condition for Phalloceros (Fig. S6). In males, a gonopodium with two outward positioned hooks on gonopodium paired appendix is inferred as the ancestral state for the genus. The presence of hooks in an inward position on both halves on symmetric paired appendix is result of a single evolutionary transition, coinciding with the shift in the female urogenital papilla turned left. In addition to four terminal shifts from two hooks in the outward position to single hook on the left half (e.g., P. buckupi, P. spiloura, P. lucenorum and P. reisi), the reconstruction based on the concatenated tree also inferred an ancestral transition from two hooks in the outward position to single hook in clade C (Fig. S7). Two events of hooks lost occurred in species that have hooks on both halves outward as ancestral. It is noteworthy that transitions in the gonopodium hooks are accompanied by shifts in the female urogenital papilla shifts (i.e., changes in state occur in concert) at least three times, including an evolutionary reversal to the ancestral states for both traits in clade C (Fig. 3). In terms of the lateral spot pattern observed in the lateral of body (Fig. 1, S8 and Table 1) that is hypothesized to play a role in mate recognition, an elliptical shaped spot is the ancestral condition in Phalloceros. Shifts to different lateral spot shapes, or spot loss, occurred in several species and mostly among terminals within the phylogeny, with only a single case of a shared state between two closely related species (i.e. round shape of the lateral spot in P. occelatus and P. cf. mikromattos).

3.4. Species distributions Phalloceros diversity and high level of sympatry is mainly observed in basins on the eastern coast of Brazil (Fig. 2), especially in tributaries of the Paranaguá and Babitonga estuaries, and in small streams along the coast of São Paulo and Rio de Janeiro states (around Ubatuba and Paraty

12

municipalities). Most co-occurring species tend to be non-sister taxa, with the exception of the sympatrically sister-pairs P. buckupi and P. megapolos distributed in tributaries of the Babitonga Bay, and P. reisi and P. harpagos in the Upper Tietê (although sister relationships for the later pair of sister taxa is supported only in the individual-based tree estimates; Figs. S2 and S3). Currently, out of the 22 species described for Phalloceros, only eight are strictly allopatric (Table 1). Most of these allopatric species are limited to the northern peripheral portions of distribution for the genus and three of them are endemic to small river basins along the coast (P. anisophallos, P. aspilos and P. enneaktinos; Fig. 2a). All the remaining 14 species (plus P. sp. L and P. sp. R) are found in sympatry with at least one other species of this genus (Table 1 and 2). Three species – P. caudimaculatus, P. spiloura and P. harpagos – have broad geographic distributions and co-occur with many other species (i.e., with two, six and seven other species, respectively; Table 1 and 2). In a few cases of distributional overlap, species occupy different portions of the same river basin (e.g. P. caudimaculatus and P. heptaktinos inhabit the lower versus headwaters of the Arroio dos Ratos River basin, respectively; there is spatial separation of P. harpagos, P. reisi and P. lucenorum in the Ribeira do Iguape River basin), but in most cases sympatric species occur in the same locality (syntopy; Table 1 and S3), especially within the coastal drainages of Southeastern Brazil. For example, there are several cases of sympatry between P. buckupi, P. pellos, P. titthos, and P. megapolos in the tributaries of Babitonga and Guaratuba bays. There is a strong geographic structure of the three clades. Clade A has the largest number of species in allopatry (i.e., three or four species, depending on the phylogenetic reconstruction) and is distributed in the north towards Paraíba do Sul and the coastal drainages of Rio de Janeiro, Espírito Santo e Bahia states in Brazil (e.g. P. enneaktinos, P. elachistos and P. ocellatus; Fig. 2

13

and Table 1). In contrast, all species within clade B occur in sympatry with other Phalloceros species and are mostly distributed in southern drainages along the coast and inland (e.g., Uruguay and Iguaçu river basins; Fig. 2). Species in clade C are distributed mostly in central coastal basins (i.e., between Paraíba do Sul River basin and Paranaguá Bay), except for P. uai, which is allopatrically distributed in the headwaters of São Francisco River basin.

3.5. Morphological characters and sympatry There are 22 occurrences of sympatry among species of Phalloceros and in all cases sympatric species differ at some morphological character mapped here (Table 2). For example, there is at least one morphological difference related to sexual characters between the species in 20 out of the 22 cases of sympatry: only female papillae (six occurrences), only males gonopodium (seven) or both combined (seven). Unlike the sexual characters, there is less differentiation in the color pattern (lateral spot) among sympatric species, with differences observed in half of sympatric species and is primarily the result of the unique presence of an extra spot on the caudal peduncle of P. spiloura.

4. DISCUSSION Our study gives insights into the evolutionary processes leading to diversification of Phalloceros and into the high levels of sympatry between its species. Specifically, diversification along the coastal Brazilian drainages in Phalloceros is mostly characterized by co-occurrence of non-sister taxa, and concerted variation in male and female sexual traits, suggesting a role of sexual traits (e.g., prezygotic mechanisms) in the divergence process. These findings highlight the potential of

14

future directions to understand microevolutionary processes associated with the evolution of reproductive isolation in distinct biogeographical contexts.

4.1. Phylogenetic relationships and species diversity Phylogenetic estimates based on the genomic data are, to some degree, congruent with a previous analysis based on morphology (Lucinda and Reis, 2005; Lucinda, 2008), but differ in aspects that suggest previous relationships may have been misled by convergent evolution of some sexual traits. The morphologically based hypothesis of species relationships of Phalloceros was built upon characters of osteology, soft anatomy and coloration, but also highlighted the importance of genital structures (Lucinda and Reis, 2005; Lucinda, 2008). This set of characters is important for Poeciliidae systematics at various levels of classification (e.g., inter- and intrageneric; Rosen and Bailey, 1963; Parenti, 1981), and congruence with some aspects of the genetic based phylogenetic estimates corroborate proposed relationships based on patterns of character evolution. For example, the close relationships of P. aspilos, P. tupinamba and P. sp. L (and likely P. leptokeras, but missing in our hypothesis) recovered with our genetic data is also supported by homoplasy-free derived structures on both male and female genitalia (Figs. 1 and 3; Lucinda, 2008: clade 81). However, sexual characters may also be subject to convergent evolution (Jones et al., 2016), which could potentially lead to disagreement between morphology and our molecular based hypotheses. For example, in contrasts to the monophyletic set of all species with right-turned urogenital papilla proposed by the morphological inference (Fig. 1; Lucinda, 2008: clade 76), our phylogenetic estimate based on loci from across the genome suggests that a right-turned papilla is an evolutionary labile, and specifically, was loss two times in clades B and C (Fig. 3). Similarly, the morphological based phylogeny suggested a

15

monophyletic set of taxa with a hook in the left half of the gonopodium appendix (i.e., the species P. lucenorum, P. spiloura, P. reisi, P. anisophallos, P. pellos, P. buckupi and P. uai; Fig. 1; Lucinda, 2008: clade 74). However, only some of these taxa are estimated to be closely related in our phylogenetic analyses, with P. spiloura and P. buckupi being more closely related to taxa with hooks in both halves of the paired gonopodium appendix (Figs. 3, S2 and S3). It is interesting to note that both P. spiloura and P. buckupi possess similar sized appendices as other species in clade B even though they have a single hook in the paired appendix. Therefore, the presences of single hook on left appendix half and the paired appendix size asymmetry are likely two independent transitions. Sources of disagreement between genetic and morphologically based estimates are not limited to genital traits. For example, our phylogenetic estimates suggest that an elliptical shaped lateral spot is the ancestral state of the genus (Fig. S8), in contrast to the proposed lack of a lateral spot in the morphological hypothesis (Lucinda, 2008: clade 89). A diversity of different shapes of the lateral spots across the tips of the phylogeny also shows the lability of this character and that it retains relative little phylogenetic signal in this genus. Although the sampling is somewhat limited within each species, and caution with interpreting genetic data alone (Sukumaran and Knowles 2017), most species form monophyletic lineages (i.e., individuals within a species share a most recent common ancestor with each other), corroborating morphological based proposed species boundaries. Given that Phalloceros species exhibit high degrees of sympatry with congeners (Fig. 2 and Table 2), the genetic exclusivity of species lineages supports the hypothesis of reproductive isolation (Mayr, 1963) among the taxa. The general exception to the monophyly of taxa is P. harpagos, and it has been the subject of speculation regarding potential cryptic diversity. According to Lucinda (2008), variation in

16

morphological features in this species, such as color pattern and size of the hooks on gonopodial appendix, has been found among geographically distinct populations. Ecomorphological differences have also been described between populations in different drainages (Leitão et al., 2018). Our data supports that such morphological variance may be indicative of additional species, with P. harpagos represented by at least three distinct lineages (Fig. 3) that are more closely related to species in close geographic areas than to its putative conspecifics. The different lineages of P. harpagos may represent distinct species, given divergence previous noted in male genitalic traits (Lucinda, 2008), reinforcing the role of these characters in the speciation process. On the other hand, the polyphyly of P. harpagos could also be a result of gene flow (i.e., hybridization and introgression) with other co-distributed taxa over time. The retention in P. harpagos of the ancestral states of Phalloceros for the sexual traits makes these two scenarios difficult to distinguish without further analyses. However, the potential of undiscovered diversity is not limited to P. harpagos. The morphotypes Phalloceros sp. R and Phalloceros sp. L suggest undescribed diversity in populations inhabiting small coastal streams in São Paulo State, Brazil.

4.2. Morphological divergence and diversification Males of the family Poeciliidae show a wide variety of traits associated with sexual selection, from differing levels of sexual dichromatism to elaborate ornaments, with Phalloceros males characterized by a long gonopodium and a smaller body size than females. These traits are commonly found in poeciliids (Parenti, 1981) and are known to facilitate sneak or coercive mating strategies (Pollux et al., 2014). Phalloceros is one of the exceptions in poeciliids (along with Pamphorichthys) in that females lack superfetation (i.e., they carry a single brood rather than embryos fertilized at different times; Arias and Reznick, 2000; Pollux et al., 2014). Our

17

results show that not only have male genitalic characters undergone a number of morphological transitions, but they have also evolved in concert with changes in female genitalic characters (Fig. 3), suggesting a role for sexual selection in the diversification of this group. However, given that most co-distributed species are not sister taxa (discussed below), divergence driven by sexual selection may be more of a consequence of differences accumulated in geographic isolation, rather than the driver of speciation per se. It is noteworthy that in the two instances of sympatric sister-species (i.e., P. megapolos + P. buckupi, and potentially P. reisi + P. harpagos based on the concatenated RAxML and individual SVDquartets reconstructions), divergence in female papilla and male gonopodium, as well as the color pattern on the lateral portion of body in the latter pair, suggest a role for sexual selection in species divergence that is not predicated on initial divergence in geographic isolation. In poeciliids lateralization during sexual courtship is known in several species, with dextral males being only able to mate with sinistral females and vice-versa (e.g., Garman, 1896; Rosen and Bailey, 1963; Miller, 1979). Bias in the angle is also reported in individuals with symmetrical sexual features, demonstrating that lateralization preference may be present even in the absence of structural differences (Bisazza et al., 1998). Some species of poeciliids (including Phalloceros species) have these traits fixed within species, suggesting that lateralization might play an important role during Phalloceros reproduction. Little intraspecific variation in the female papilla direction and male gonopodium hooks were reported for the species of Phalloceros (except for P. harpagos; Lucinda, 2008; Ono and Shibatta, 2015). Here we also observed intermediate states in some species. For example, differences in the direction of the papilla as directed slightly right, instead of straight in some populations of P. caudimaculatus. If

18

these differences may be a response to co-existence with other Phalloceros species (i.e., a type of character displacement) or just standing phenotypic variation needs to be further investigated. Furthermore, differences in phenotypic characters of co-distributed species also suggests a pattern of reproductive character displacement (Anderson and Langerhans, 2015; RothMonzón et al., 2017); whether this pattern is caused by mechanisms associated with reinforcement (i.e., to avoid the production of unfit hybrids) or some other process is not clear. Irrespective of the exact process underlying the observed pattern, a strong mismatch in sexual traits among sympatric taxa suggests that co-existence may be linked to divergent sexual traits that maintain species genetic distinctiveness through mechanical disruptions of interbreeding. Asymmetry observed in genitalia, especially in the direction of female’s papilla, could restrict gonopodial contact and, consequently, decrease the success of sneak or coercive mating behavior by males (Brennan et al., 2009) among co-distributed conspecifics. Between non-sister taxa, the sexual trait mismatch occurring in 20 out of 22 instances of sympatry suggests that this pattern is not random (Table 2). Sexual selection may not be the only force involved in species divergence, as with other poeciliids, but little work has been done on Phalloceros ecologies and predation regimes (Endler, 1982). Among Phalloceros species, taxa with more conspicuous lateral spots (highly pigmented rounded or squared) are interestingly restricted to allopatric species (e.g., the ocellus-like lateral spot of P. ocellatus and P. mikromatos, or the uniquely squared shape lateral spot in P. uai; Fig. 1). Ocellus-like spot are typically associated with predation avoidance within fish groups (Neudecker 1989; Kelley et al., 2013) and therefore these traits may reflect relaxation of selection on characters associated with species recognition relative to those in sympatric species pools. Within poeciliids, divergent natural and sexual selection has been largely evoked to

19

explain differences in phenotypes (summarized in Seehausen and Wagner, 2014). Further investigation is required to understand the extent to which the differences reflect varying strengths of natural and sexual selection in sympatry and allopatry in Phalloceros, with the intriguing possibility that interactions at the community level (e.g., predators) may be affecting how selection acts on Phalloceros populations.

4.3. Geography of diversification In the Neotropics, geographic isolation following dispersal events among drainages has been an explanation for the high diversity of freshwater fishes (Lundberg et al., 1998; Albert et al., 2011). Dispersal between isolated drainages is often associated with temporary connections forged during sea-level retreat and/or river captures. Phalloceros is member of a family of freshwater fishes (Poeciliidae) that often show some tolerance to salinity and are frequently found in brackish water (but not in saltwater; Lucinda, 2003), being collected is streams near shorelines. This suggests dispersal in coastal rivers by paleodrainages, which connected currently isolated rivers in periods of sea level retreat during the Pleistocene (Weitzman and Weitzman, 1988; Thomaz et al., 2015, 2017), may have served as a source of dispersal and helps explain the broad distribution of some of the coastal Phalloceros species (Fig. 2). On the other hand, Phalloceros species are sometimes the only members of the fish fauna distributed in both primary and secondary order streams. They are often represented among the relatively speciespoor community that often occurs in upper reaches of river basins (Sabino and Côrrea e Castro, 1990; Esteves and Lóbon-Cerviá, 2001), suggesting that headwater capture events in these watersheds (Ribeiro et al., 2006; Lima et al., 2017) may have also played a role in dispersing Phalloceros taxa between neighboring basins. Such mechanisms of dispersal likely have been

20

involved throughout their evolutionary history because many of the co-distributed species are not sister taxa and therefore represent cases of secondary sympatry. Viviparity is often associated with high dispersal success and lower extinction rate in poeciliids. The potential establishment of single gravid females, into new geographic regions may facilitate speciation in the group (Meyer and Lideard, 1993; Mank and Avise, 2006). This outcome may be restricted to fragmented landscapes, such as rivers (Helmstetter et al., 2016), since large dispersal capability in homogeneous environments (e.g., terrestrial, lakes and marine) would be responsible for genetic homogenization. However, it is interesting to note that despite support for high dispersal potential evidenced by the high co-occurrence of non-sister taxa, there is nonetheless large number of endemic Phalloceros species with restricted distributions. Among taxa that have diversified within a basin (e.g., P. buckupi and P. megapolos), sympatric speciation in the face of gene flow (Coyne and Orr 2004) or allopatric speciation in different rivers or river stretches followed by secondary contact are potential explanations. Independent of the speciation process, the unique shape of the male gonopodium in P. megapolos (i.e., wing like projections gonopodium; Lucinda, 2008) may have been responsible for prezygotic mechanism of isolation between these two species. Whether this divergence was the driver of speciation, or a consequence of speciation (i.e., a divergence that occurred after the origin of species in some other context) remains an open question. Future study of sympatric and allopatric Phalloceros taxa will be needed before understanding how these processes of diversification might differ depending on the geographic context of divergence.

4.4. Conclusions

21

Our genomic data provide relatively well-resolved species relationships that shed light on potential evolutionary processes during the speciation and diversification of Phalloceros, and specifically, the geography of divergence and possible role of sexual selection. The high level of sympatry and genetic exclusivity of morphologically defined species demonstrate the potential that this genus has for studying genetic, morphological and behavioral differentiation that may lead to speciation. With most sympatry limited to non-sister taxa, co-occurrence in this case is consistent with allopatric speciation, followed by secondary sympatry. With concerted divergence of male and female genitalic traits, speciation in this genus could have been also mediated by strong sexual selection. Future research in this genus focusing on aspects of genital functional morphology, ecology and behavior at local scales are necessary to disentangle which processes (i.e., neutral processes, sexual and/or natural selection) played a major role in Phalloceros diversification.

22

5. ACKNOWLEDGEMENTS For loans of samples we thank C. Lucena and M. Lucena (MCP); and J. Wingert (UFRGS). We thank also D. Nelson and D. O’Foighil for help with the MTA and loan of material to UMMZ. Thanks to B. Sidlauskas for assistance in analyses. ATT thanks Rackham International Research Award - University of Michigan for financial support for the development of this study. TPC acknowledges his support from a postdoctoral fellowship of PNPD-CAPES. LRM acknowledges CNPq (processes # 307890/2016-3 and 401204/2016-2). Thanks to J. Wingert for help with photos of genitalia.

6. REFERENCES Albert J.S., Petry P., Reis R.E., 2011. Major biogeographic and phylogenetic patterns. In: Albert J.S., Reis R.E. (Eds.), Historical biogeography of Neotropical freshwater fishes. University of California Press, Berkley, pp. 21-57. Anderson C.M., Langerhans R.B., 2015. Origins of female genital diversity: Predation risk and lock‐ and‐ key explain rapid divergence during an adaptive radiation. Evolution 69, 24522467. Arias A.L., Reznick D., 2000. Life history of Phalloceros caudimaculatus: a novel variation on the theme of livebearing in the family Poeciliidae. Copeia 2000, 792-798. Bisazza A., Rogers L.J., Vallortigara G., 1998. The origins of cerebral asymmetry: a review of evidence of behavioural and brain lateralization in fishes, reptiles and amphibians. Neurosci. Biobehav. Rev. 22, 411-426.

23

Brennan P.L., Clark C.J., Prum R.O., 2009. Explosive eversion and functional morphology of the duck penis supports sexual conflict in waterfowl genitalia. Proc. R. Soc. London B. 1686, 1309-1314. Chifman J, Kubatko L. 2014. Quartet inference from SNP data under the coalescent model. Bioinformatics 30, 3317-3324. Coyne J.A., Orr H.A, 2004. Speciation. Sinauer, Sunderland. Crispo E., Bentzen P, Reznick D.N., Kinnison M.T., Hendry A.P., 2006. The relative influence of natural selection and geography on gene flow in guppies. Mol. Ecol 15, 49-62. Eaton D.A., 2014. PyRAD: assembly of de novo RADseq loci for phylogenetic analyses. Bioinformatics 30, 1844-1849. Eigenmann C.H., 1907. The Poeciliid Fishes of Rio Grande do Sul and the La Plata Basin. Smithsonian Institution, Washington. 32, 1532. Endler J.A., 1982. Convergent and divergent effects of natural selection on color patterns in two fish faunas. Evolution 36, 178-188. Endler J.A., 1983. Natural and sexual selection on color patterns in poeciliid fishes. Env. Biol. Fish. 9, 173-190. Esteves K.E., Lobón-Cerviá J., 2001. Composition and trophic structure of a fish community of a clear water Atlantic rainforest stream in southeastern Brazil. Env. Biol. Fish. 62, 429-440. Fricke R., Eschmeyer W.N., Fong J.D., 2018. SPECIES BY FAMILY/SUBFAMILY. (http://researcharchive.calacademy.org/research/ichthyology/catalog/SpeciesByFamily.asp). Electronic version accessed Nov. 2018. Froese R., Pauly D., 2017. FishBase: World Wide Web electronic publication. http://www.fishbase.org (accessed October 2017)

24

Garman S., 1896. Cross fertilization and sexual rights and lefts among vertebrates. Amer. Nat. 30, 232. Helmstetter A.J., Papadopulos A.S., Igea J., Van Dooren T.J., Leroi A.M., Savolainen V., 2016. Viviparity stimulates diversification in an order of fish. Nature Comm. 7, 11271. Hensel R., 1868. Beiträge zur Kenntniss der Wirbelthiere Südbrasiliens. (Fortsetzung). Archiv für Naturgeschichte 34, 323-375. Hrbek T., Seckinger J., Meyer A., 2007. A phylogenetic and biogeographic perspective on the evolution of poeciliid fishes. Mol. Phyl. Evol. 43, 986-998. Jennions M.D., Kelly C.D., 2002. Geographical variation in male genitalia in Brachyrhaphis episcopi (Poeciliidae): is it sexually or naturally selected? Oikos 97, 79-86. Jones J.C., Fruciano C., Keller A., Schartl M., Meyer A., 2016. Evolution of the elaborate male intromittent organ of Xiphophorus fishes. Ecol Evol. 6, 7207-7220. Kallman K.D., Walter R.B., Morizot D.C., Kazianis S., 2004. Two new species of Xiphophorus (Poeciliidae) from the Isthmus of Tehuantepec, Oaxaca, Mexico, with a discussion of the distribution of the X. clemenciae clade. Am. Mus. Nov. 3441, 1-34. Kelley J.L., Fitzpatrick J.L., Merilaita S., 2013. Spots and stripes: ecology and colour pattern evolution in butterflyfishes. Proc. R. Soc. B. 280, 20122730. Langerhans R.B., Layman C.A., DeWitt T.J., 2005. Male genital size reflects a tradeoff between attracting mates and avoiding predators in two live-bearing fish species. PNAS. 102, 76187623. Langerhans R.B., Gifford M.E., Domínguez‐ Domínguez O., García‐ Bedoya D., DeWitt T. J., 2012. Gambusia quadruncus (Cyprinodontiformes: Poeciliidae): a new species of mosquitofish from east‐ central México. J. Fish Biol. 81, 1514-1539.

25

Leitão M.L.C, Lima I., Hiroki K.A.N., Pelli A, de Souza F., 2018. Diferenças ecomorfológicas e funcional em populações de Phalloceros harpagos Lucinda, 2008, na Bacia do alto rio Paraná. Biota Amazônia 8: 34-38. Li H., Durbin R., 2009. Fast and accurate short read alignment with Burrows-Wheeler Transform. Bioinformatics 25, 1754-60. Lima S.M.Q., Berbel-Filho W.M., Araújo T.F.P., Lazzarotto H., Tatarenkov A., Avise J.C. (2017). Headwater capture evidenced by paleo-rivers reconstruction and population genetic structure of the armored catfish (Pareiorhaphis garbei) in the Serra do Mar mountains of southeastern Brazil. Front. Gen. 8, 1-8. Lucinda P.H.F., Reis R.E., 2005. Systematics of the subfamily Poeciliinae Bonaparte (Cyprinodontiformes: Poeciliidae), with an emphasis on the tribe Cnesterodontini Hubbs. Neotrop. Ichthyol. 3, 1-60. Lucinda P.H.F., de Souza Rosa R., Reis R.E., 2005. Systematics and biogeography of the genus Phallotorynus Henn, 1916 (Cyprinodontiformes: Poeciliidae: Poeciliinae), with description of three new species. Copeia 3, 609-631. Lucinda P.H., 2003. Family Poeciliidae.In: Reis R.E., Kullander S.O., Ferraris Jr. C.J. (Eds.) Check list of the freshwater fishes of South and Central America. Edipucrs, Porto Alegre pp. 555-585. Lucinda P.H.F., 2005a. Systematics and biogeography of the genus Phalloptychus Eigenmann, 1907 (Cyprinodontiformes: Poeciliidae: Poeciliinae). Neotrop. Ichthyol. 3, 373-382. Lucinda P.H.F., 2005b. Systematics of the genus Cnesterodon Garman, 1895 (Cyprinodontiformes: Poeciliidae: Poeciliinae). Neotrop. Ichthyol. 3, 259-270.

26

Lucinda P.H.F., 2008. Systematics and biogeography of the genus Phalloceros Eigenmann, 1907 (Cyprinodontiformes: Poeciliidae: Poeciliinae), with the description of twenty-one new species. Neotrop. Ichthyol. 6, 113-158. Lundberg J.G., Marshall L.G., Guerrero J., Horton B., Malabarba M.C.S.L., Wesselingh F., 1998. The stage for Neotropical fish diversification: a history of tropical South American rivers. In: Malabarba L.R., Reis R.E., Vari R.P., Lucena Z.M.S., Lucena C.A.S. (Eds.), Phylogeny and classification of Neotropical fishes. Edipucrs, Porto Alegre, pp. 13-48. Mank J.E., Avise J.C., 2006. Supertree analyses of the roles of viviparity and habitat in the evolution of atherinomorph fishes. J. Evol. Bio. 19, 734-740. Mayr E., 1963. Animal species and evolution. Cambridge (MA): Harvard University Press. Meyer A., Lydeard C., 1993. The evolution of copulatory organs, internal fertilization, placentae and viviparity in killifishes (Cyprinodontiformes) inferred from a DNA phylogeny of the tyrosine kinase gene X-src. Proc. R. Soc. B. 254, 153-162. Miller R.R., 1979. Ecology, habits and relationships of the middle american cuatro ojos, Anableps dowi (Pisces: Anablepidae). Copeia 1, 82-91. Murphy W.J., Eizirik E., O'brien S.J., Madsen O., Scally M., Douady C.J., Teeling E., Ryder O.A., Stanhope M.J., de Jong W.W., Springer, M. S., 2001. Resolution of the early placental mammal radiation using Bayesian phylogenetics. Science 294, 2348-2351. Neudecker S., 1989. Eye camouflage and false eyespots: chaetodontid responses to predators. Environ. Biol. Fish. 25, 143 – 157. Nosil P., Gompert Z., Farkas T.E., Comeault A.A., Feder J.L., Buerkle C.A., Parchman, T.L., 2012. Genomic consequences of multiple speciation processes in a stick insect. Proc. R. Soc. B. rspb20120813

27

Oneal E., Knowles L.L., 2013. Ecological selection as the cause and sexual differentiation as the consequence of species divergence? Proc. R. Soc. B. 280, 20122236. Ono R.H., Shibatta O.A., 2015. Avaliação de caracteres taxonômicos diagnósticos do aparelho genital de Phalloceros harpagos Lucinda, 2008 (Cyprinodontiformes: Poeciliidae). Boletim da Sociedade Brasileira de Icitiologia 115, 22-25. Paradis E., Claude J., Strimmer K., 2004. APE: analyses of phylogenetics and evolution in R language. Bioinformatics 20: 289-290. Parenti L.R., 1981. A phylogenetic and biogeographic analyses of Cyprinodontiform fishes (Teleostei, Atherinomorpha). Bull. Amer. Mus. Nat. Hist. 168, 341-557. Peterson B.K., Weber J.N., Kay E.H., Fisher H.S., Hoekstra H.E., 2012. Double digest RADseq: an inexpensive method for de novo SNP discovery and genotyping in model and non-model species. PloS one 7, e37135. Pollux B.J.A., Pires M.N., Banet A.I., Reznick D.N., 2009. Evolution of placentas in the fish family Poeciliidae: an empirical study of macroevolution. Annu. Rev. Ecol. Evol. Syst. 40, 271-289. Pollux B.J.A., Meredith R.W., Springer M.S., Garland T., Reznick D.N., 2014. The evolution of the placenta drives a shift in sexual selection in livebearing fish. Nature 513, 233-236. R Core Team. R: A language and environment for statistical computing, 2017. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/ Reis R.E., Albert J.S., Di Dario F., Mincarone M.M., Petry P., Rocha L.A., 2016. Fish biodiversity and conservation in South America. J. Fish Biol. 89, 12-47.

28

Ribeiro A.C., Lima F.C., Riccomini C., Menezes N.A., 2006. Fishes of the Atlantic Rainforest of Boracéia: testimonies of the Quaternary fault reactivation within a Neoproterozoic tectonic province in Southeastern Brazil. Ichthyol. Expl. Fresh. 17, 157-164. Ribeiro A.C., 2006. Tectonic history and the biogeography of the freshwater fishes from the coastal drainages of eastern Brazil: an example of faunal evolution associated with a divergent continental margin. Neotrop. Ichthyol. 4, 225-246. Rosen D.E., Bailey R.M., 1963. The poeciliid fishes (Cyprinodontiformes): their structure, zoogeography, and systematics. Bulletin of the AMNH. 126, 1-176. Roth-Monzón A.J., Scott L.E., Camargo A.A., Clark E.I., Schott E.E., Johnson J.B., 2017. Sympatry predicts spot pigmentation patterns and female association behavior in the livebearing fish Poeciliopsis baenschi. PloS one 12, e0170326. Sabino J., Corrêa e Castro R.M., 1990. Feeding biology, activity period and spatial distribution of fishes in an Atlantic forest stream in southeastern Brazil. Rev. Bras. Biol. 50, 23-36. Sanderson M.J., 2002. Estimating absolute rates of molecular evolution and divergence times: a penalized likelihood approach. Mol. Biol. Evol. 19: 101-109. Schartl M., Walter R.B., Shen Y., Garcia T., Catchen J., Amores, A., Braasch I., Chalopin D., Volff J.N., Lesch K.P., Bisazza A., Minx P., Hillier L. Wilson R.K., Fuerstenberg S., Boore J., Searle S., Postlethwait J.H., Warren W.C., 2013. The genome of the platyfish, Xiphophorus maculatus, provides insights into evolutionary adaptation and several complex traits. Nature Genet. 45, 567-572. Seehausen O., Wagner C.E., 2014. Speciation in freshwater fishes. Annu. Rev. Ecol. Evol. Syst. 45, 621-651.

29

Stamatakis A., 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312-1313. Sukumaran J., Knowles L.L., 2017. Multispecies coalescent delimits structure, not species. PNAS. 114, 1607-1612. Swofford D.L., 2003. PAUP*: phylogenetic analysis using parsimony and other methods, version 4.0 b10. Sinauer Associates, Sunderland. Thomaz A.T., Malabarba L.R., Bonatto S.L., Knowles L.L., 2015. Testing the effect of palaeodrainages versus habitat stability on genetic divergence in riverine systems: study of a Neotropical fish of the Brazilian coastal Atlantic Forest. J. Biogeogr. 42, 2389-2401. Thomaz A.T., Malabarba L.R., Knowles L.L., 2017. Genomic signatures of paleodrainages in a freshwater fish along the southeastern coast of Brazil: genetic structure reflects past riverine properties. Heredity 119, 287-294. Thomaz, A.T., Carvalho T.P., Malabarba L.R., Knowles L.L, 2018. Geographic distributions, phenotypes, and phylogenetic relationships of Phalloceros (Cyprinodontiformes: Poeciliidae): insights about diversification among sympatric species pools [DATA SET]. University of Michigan Deep Blue Data Repository. Available from: https://doi.org/10.7302/Z2G44NJ2. Vari R.P., Malabarba, L.R., 1998. Neotropical ichthyology: an overview. Phylogeny and classification of Neotropical fishes. In: Malabarba L.R., Reis R.E., Vari R.P., Lucena Z.M.S., Lucena C.A.S. (Eds.), Phylogeny and classification of Neotropical fishes. Edipucrs, Porto Alegre, pp 1-12. Wagner C.E., Keller I., Wittwer S., Selz O.M., Mwaiko S., Greuter L., Sivasundar A., Seehausen O., 2013. Genome‐ wide RAD sequence data provide unprecedented resolution of species

30

boundaries and relationships in the Lake Victoria cichlid adaptive radiation. Mol. Ecol. 22, 787-798. Wei, T., Simko, V., Levy, M., Xie, Y., Jin, Y., & Zemla, J. (2017). Package ‘corrplot’. Statistician, 56, 316-324. Weitzman S.H., Menezes N.A., Weitzman M.J., 1988. Phylogenetic biogeography of the Glandulocaudini (Teleostei: Characiformes, Characidae) with comments on the distribution of other freshwater fishes in eastern and southeastern Brazil. In: Vanzolini P.E., Heyer W.R. (Eds.), Proceedings of a workshop on Neotropical distribution patterns. Academia Brasileira de Ciências, Rio de Janeiro, pp. 379-427.

31

TABLES Table 1. Focal taxa and summaries of morphological characters used to diagnose Phalloceros species and aspects of their distributions based on data collected for this study and from Lucinda (2008); species not included in this study are marked by an asterisk. Most of the sympatry are instances of co-occurrences within a river basin in the same or geographic proximate localities, however, the few cases of occurrences in the same river basin, but geographically distant (i.e., headwater and lowland portions) are indicated by two asterisks. Species

Lateral spot

1

P. alessandrae

Elliptical

Female urogenital papilla Right turned

2

P. anisophallos

Elliptical

Right turned

3

P. aspilos

Left turned

4

P. buckupi

Inconspicuous or absent Elliptical

5

P. caudimaculatus

Elliptical

Straight

Hooks on both appendix halves; halves symmetrical and slender Hook on left half of appendix; halves asymmetrical: right side wider, halves sickle like Hooks on both appendix halves (inner side), halves symmetrical Hook on left half of appendix, halves symmetrical, sickle like Hooks absent, halves symmetrical

6

P. elachistos

Straight

Hooks on both appendix halves, halves symmetrical

No

7 8

P. enneaktinos P. harpagos

Inconspicuous or absent Elliptical Elliptical

Straight Straight

Hooks on both appendix halves, halves symmetrical Hooks on both appendix halves, halves symmetrical

9 10

P. heptaktinos* P. leptokeras*

Absent Rectangular

Straight Left turned

11 12

P. leticiae* P. lucenorum

Rounded Elliptical

Straight Right turned

13 14

P. malabarbai P. megapolos

Elliptical Elliptical

Right turned Right turned

Hooks absent, halves symmetrical Hooks on both appendix halves (inner side), halves symmetrical Hooks absent, halves symmetrical Hook on left half of appendix; halves asymmetrical: right side wider, halves sickle like Hooks on both appendix halves, halves symmetrical Hooks on both appendix halves, halves symmetrical and with wing-like projections

15 16 17

P. mikrommatos P. ocellatus P. pellos

Rounded Rounded Elliptical

Straight Straight Right turned

Right turned

Male gonopodium

Hooks absent, halves symmetrical Hooks absent, halves symmetrical Hook on left half of appendix; halves asymmetrical:

Sympatric taxa 17

River drainage, State

No No

Rio Parati, Barra Grande, São Roque, Taquari and Itinguçu, RJ Rio Parati-Mirim, RJ

14, 17, 20

Tributaries of Paranaguá and Babitonga Bay, PR

9**, 19

No 10, 12**, 13, 17, 18, 19, 21 5**, 19 8, 18**

Laguna dos Patos, rio Uruguai, and coastal drainages of RS and SC in Brazil, Uruguay and Argentina Rio Doce, Santa Maria da Vitória, Jucu, Timbuí and coastal drainages, ES Córrego da Toca do Boi, RJ Rio Paraná-Paraguay basin and coastal drainages from rio Itabapoana to Araranguá, ES-SC Arroio dos Ratos, Jacuí basin, RS Rio Paraíba do Sul, RJ

No 8**, 18**

Rio Araguaia, GO Rio Juquiá, Ribeira de Iguape basin, SP

8, 19 4, 17, 19, 20

Rivers near Itapoá and Tijucas, SC Rio São João, Cubatão (Norte) and small adjacent drainages, which flows into the Baía de Babitonga and Guaratuba, PR Rio João de Tiba, BA Coastal tributaries between Barra Seca and Ibarube, ES-BA Tributaries of Paranaguá Bay, PR

No No 1, 4, 8, 14, 19

Tributaries of Paranaguá Bay, PR

32

18

P. reisi

Right turned

P. titthos P. tupinamba

Inconspicuous or absent Elliptical plus peduncle spot Elliptical Elliptical

19

P. spiloura

20 21 22

P. uai

Squared

Right turned

23

P. sp. L

Elliptical

Left turned

24

P. sp. R

Elliptical

Right turned

Right turned Straight Left turned

right half wider Hook on left half of appendix; halves asymmetrical: right half wider Hooks absent, halves symmetrical Hooks on both appendix halves, halves symmetrical Hooks on both appendix halves (inner side), halves symmetrical Hook on left half of appendix; halves asymmetrical: right half wider Hooks on both appendix halves (inner side), halves symmetrical Hook on left half of appendix; halves asymmetrical: right half wider

8, 10**, 12** 5, 8, 9, 13, 14, 17 4, 14 8

Rio Tietê, Paraíba do Sul, Ribeira de Iguape, and small coastal drainages, SP Rio Uruguay, Jacuí, Iguaçu and coastal drainages, RS-SC Tributaries of Guaratuba and Paranaguá Bays, PR Coastal rivers near Ubatuba and rio Macacu, SP-RJ

No

Rio das Velhas, São Francisco basin, MG

24

Coastal rivers near Ubatuba, SP

23

Coastal rivers near Ubatuba, SP

33

Table 2. Summary of morphological characters observed in each sympatric species pairs. Upper diagonal indicates spot pattern on the side of the body, and lower diagonal sexual characters: female urogenital papilla direction (straight, right, or left), and male gonopodium halves symmetry (as = asymmetrical; s = symmetrical) plus hook presence (in both halves, just in the left halve, or completely absent). Orange color indicates different sexual character(s), green indicates difference in spot pattern and grey depicts no difference in morphological characters in sympatry.

P. alessandrae

P. buckupi

P. caudimaculatus

P. harpagos

P. heptaktinos*

P. leptokeras* P. lucenorum

P. malabarbai

P. megapolos

P. pellos

P. reisi

P. spiloura

Absent/ Elliptical

Elliptical plus 2nd spot/ Elliptical plus 2nd spot/ Elliptical plus 2nd spot/

P. titthos

P. tupinamba

P. sp. L

P. sp. R

Spot pattern P. alessandrae

Elliptical

P. buckupi

Elliptical

P. caudimaculatus

Rectangular/ Elliptical

P. harpagos

Straight / Left Straight / Right Straight / Right

P. leptokeras* P. lucenorum P. malabarbai

P. pellos

Left-s/ Right Both-s Both-s/ Left-s/ Right Right Left-as Left-as

P. reisi P. spiloura P. titthos P. tupinamba

Elliptical

Elliptical

Elliptical

Elliptical

AbsentStraight s

P. heptaktinos*

P. megapolos

Elliptical

Absent/ Elliptical

Absent/ Rectangular Absent/ Elliptical

Both-s Both-s/ Left-as Both-s Elliptical

Straight / Right Straight / Right Straight Absent- Straight / Right s / Right

Both-s/ Left-as Both-s/ Left/ Both-s/ Right Left-as Left-as Right Left-as Both-s/ Straight AbsentAbsent- / Right s

Right/ Left-s/ Straigh Both-s

Elliptical

Right

Right

Both-s/ Left-as

Elliptical plus 2nd spot/ Elliptical plus 2nd spot/ Elliptical plus 2nd spot/

Elliptical

Both-s/ Both-s/ Left-as/ Right Right AbsentAbsentAbsentRight/ Both-s Straigh

Straight Both-s / Left

P. sp. L

Elliptical Left/ Both-s/ Right Left-as

P. sp. R

Sexual characters: female papilla position | male hooks

34

FIGURE LEGENDS Figure 1. Species relationships of Phalloceros based on the analyses of morphological data (modified from Lucinda and Reis, 2005, and Lucinda, 2008) and plate showing representative species of Phalloceros. Note interspecific diversity of color pattern on the lateral portion of body. Males on the left and females on the right. A: P. caudimaculatus, UFRGS 22638 (Male: 20.1 mm SL; Female: 31.4 mm SL); B: P. occelatus UFRGS 11012 (Male: 17.9 mm SL; Female: 21.1 mm SL); C: Phalloceros tupinamba, UFRGS 10216 (Male: 27.7 mm SL; Female: 36.8 mm SL); D: P. spiloura UFRGS 20216 (Male: 28.6 mm SL; Female: 22.1 mm SL); E: Phalloceros reisi UFRGS 18020 (Male: 27.2 mm SL; Female: 31.7 mm SL). SL= Standard length.

Figure 2. Distribution map for the 22 valid species of Phalloceros and the two putative undescribed species used in this study. Inset (a) shows the coast of São Paulo and Rio de Janeiro states, and (b) the Paranaguá and Babitonga bays in Paraná and Santa Catarina states, which are areas that have several endemic species of Phalloceros. Note that not all overlaps are instances of sympatry, for precise information about species co-occurrences refer to Table 1.

Figure 3. Estimate of the species tree that takes into account coalescent variation among loci, based on an analysis of 18,122 SNPs using SVDquartets (Chifman and Kubatko, 2014) – for full species tree with bootstrap values see Supplementary Figure S4. Squares on the terminals are color-coded according the direction of female urogenital papilla (left) and, direction and number of hooks on gonopodium appendix (right), letter “A” within

35

red squares indicate asymmetric size of the paired appendix (others are symmetric). Arrows in male gonopodium photographs indicate the position and number of hooks. Male gonopodium from top to bottom correspond to P. caudimaculatus (UFRGS 22638; 18.2 mm SL); P. harpagos (UFRGS 20501; 23.5 mm SL); P. anisophallos (UFRGS 10250, 17.5 mm SL) and P. tupinamba (UFRGS 10216; 22.8 mm SL). Female urogenital papillae from top to bottom correspond to P. harpagos (UFRGS 20501; 35.2 mm SL); P. anisophallos (UFRGS 10250; 28.4 mm SL); P. tupinamba (UFRGS 10216 35.8 mm SL). SL = Standard length. Images of male gonopodium and female urogenital papillae were done using a stereomicroscope with a Nikon AZ100M camera attached.

36

P. mikrommatos P. leticiae P. heptaktinos P. caudimaculatus (A) P. leptokeras P. aspilos P. tupinamba (C) P. elachistos P. enneaktinos P. titthos P. occelatus (B) P. harpagos P. megapolos P. malabarbai P. alessandrae P. spiloura (D) P. reisi (E) P. anisophallos P. pellos P. uai P. buckupi P. lucenorum Phalloceros mikrommatos

Phalloceros leticiae

Phalloceros heptaktinos

Phalloceros caudimaculatus

Phalloceros leptokeras

(A)

Phalloceros aspilos

Phalloceros tupinamba

Phalloceros elachistos

Phalloceros enneaktinos

(B)

Phalloceros titthos

Phalloceros occelatus

Phalloceros harpagos

Phalloceros megapolos

(C)

Phalloceros malabarbai

Phalloceros alessandrae

Phalloceros spiloura

Phalloceros reisi

Phalloceros anisophallos

(D)

+hsu B tlu a f eD no d es ab ,d e tidE

Phalloceros pellos

Phalloceros uai

Phalloceros buckupi

Phalloceros lucenorum

(E)

y t ra Pa a ub t ba U Babi t onga

A A A A A A

SUPPLEMENTARY MATERIAL Table S1. Samples information such as collection housing, geographic location (basin), and geographic coordinates. Table S2. Pre- and post-processing statistics from ipyrad per individual. Table S3. Comparison between models of discrete character evolution of selected morphological features in Phalloceros. Table S4. List of geographic coordinates used to estimate species distributions.

Figure S1. Number of loci shared between samples based on the final dataset with 18,122 loci. Larger and darker circles indicate more shared loci between these two samples. Figure S2. Estimate of phylogenetic relationships among all samples based on concatenation of 18,122 loci. Maximum likelihood from an analysis conducted in RAxML (Stamatakis, 2014) with 1,000 bootstraps, which are shown in each node. Figure S3. Estimate of the phylogenetic relationships among all samples that takes into account genealogical discordancy among loci, based on an analysis of 18,122 SNPs using SVDquartets (Chifman and Kubatko, 2014) with 1,000 bootstraps, which are shown in each node. Figure S4. Species tree estimate that takes into account genealogical discordancy among loci, based on an analysis of 18,122 SNPs using SVDquartets (Chifman and Kubatko, 2014) with 1,000 bootstraps, which are shown in each node. Figure S5. Estimate of the phylogenetic relationships based on a single individual per species with the objective to infer the position of the root with Xyphophorus maculatus as

37

outgroup. Analysis was performed with SVDquartets (Chifman and Kubatko, 2014) with 1,000 bootstraps based on 9,444 SNPs that were mapped into X. maculatus genome. Figure S6. Maximum likelihood ancestral state reconstruction for the female urogenital papilla. Figure S7. Maximum likelihood ancestral state reconstruction for the male gonopodium. Figure S8. Maximum likelihood ancestral state reconstruction for the lateral spot pattern.

38

Highlights: - Estimates of phylogenetic relationships in Phalloceros. - Monophyly of most morphological delimited species corroborated by molecular phylogeny. - Shifts in the female urogenital papilla and male gonopodium occurred in concert. - Strong mismatch in sexual traits among sympatric taxa - Co-existence linked to divergent sexual traits that maintain species genetic distinctiveness.

39

P. tupinamba P. aspilos P. sp. L

♀ Papilla

♂ Hook

Straight

P. elachistos P. cf. mikrommatos P. ocellatus

A

Absent

P. harpagos* P. enneaktinos P. caudimaculatus P. harpagos (SC) P. malabarbai

B

Right Two outward

P. buckupi P. megapolos P. alessandrae P. spiloura P. lucenorum P. reisi

C

P. pellos

Left

Single

P. sp. R P. anisophallos P. cf. uai P. titthos P. harpagos (PR-SP) P. harpagos (Tietê)

Two inward