Analysis of the mitochondrial 12S rRNA gene supports a two-clade hypothesis of the evolutionary history of scleractinian corals

MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution 23 (2002) 137–149 www.academicpress.com

Analysis of the mitochondrial 12S rRNA gene supports a two-clade hypothesis of the evolutionary history of scleractinian corals Chaolun Allen Chen,a,* Carden C. Wallace,b and Jackie Wolstenholmeb,c a

Institute of Zoology, Academia Sinica, Nankang, Taipei 115, Taiwan Museum of Tropical Queensland, Townsville 4810, Qld., Australia Department of Marine Biology, James Cook University, Townsville 4811, Qld., Australia b

c

Received 14 June 2001; received in revised form 4 October 2001

Abstract Scleractinian corals have long been assumed to be a monophyletic group characterized by the possession of an aragonite skeleton. Analyses of skeletal morphology and molecular data have shown conflicting patterns of suborder and family relationships of scleractinian corals, because molecular data suggest that the scleractinian skeleton could have evolved as many as four times. Here we describe patterns of molecular evolution in a segment of the mitochondrial (mt) 12S ribosomal RNA gene from 28 species of scleractinian corals and use this gene to infer the evolutionary history of scleractinians. We show that the sequences obtained fall into two distinct clades, defined by PCR product length. Base composition among taxa did not differ significantly when the two clades were considered separately or as a single group. Overall, transition substitutions accumulated more quickly relative to transversion substitutions within both clades. Spatial patterns of substitutions along the 12S rRNA gene and likelihood ratio tests of divergence rates both indicate that the 12S rRNA gene of each clade evolved under different constraints. Phylogenetic analyses using mt 12S rRNA gene data do not support the current view of scleractinian phylogeny based upon skeletal morphology and fossil records. Rather, the two-clade hypothesis derived from the mt 16S ribosomal gene is supported. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Scleractinia; Mitochondrial 12S ribosomal RNA gene; Skeleton; Phylogeny

1. Introduction Skeletal formation is regarded as one of the most consistent features of the 240-million-year evolutionary history of modern scleractinian corals (Veron, 2000). Coupled with the sophisticated endosymbiotic system of dinoflagellate algae (zooxanthellae), the skeleton allows scleractinian corals to build the framework of tropical coral reefs, providing diverse habitats for the world’s diverse marine ecosystems (reviewed in Birkeland, 1997; Dubinsky, 1990). Skeletal characters, such as septa, coenosteum, thecal structure, and corallum structure, are used as fundamental features of scleractinian taxonomy (Cairns, 1999; Powers, 1970; Powers and Rholf, *

Corresponding author. Fax: +886-2-2785-8059. E-mail address: [email protected] (C.A. Chen).

1972; Vaughan and Wells, 1943; Veron, 1995; Wallace, 1999; Wells, 1956) and for construction of phylogenetic hypotheses relating to the Scleractinia (Roniewicz and Morycowa, 1993; Veron, 1995; Wallace, 1999; Wells, 1956). However, variability in skeletal morphology caused by environmental or genetic differences is frequently documented from intracolonial to interpopulation levels in scleractinian corals (reviewed in Veron, 1995), resulting in equivocal taxonomic determinations (Potts, 1984; Randall, 1976) and controversial phylogenies (Roniewicz and Morycowa, 1993; Veron, 1995). Recent analyses of DNA sequences from the mitochondrial genome of corals indicate unique molecular characteristics of gene evolution in this group of simple metazoans (Chen and Yu, 2000; Romano and Palumbi, 1996, 1997; Van Oppen et al., 1999a,b; Van Oppen et al., 2000). Characterization of a 9985-bp segment of mito-

1055-7903/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 5 5 - 7 9 0 3 ( 0 2 ) 0 0 0 0 8 - 8

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C.A. Chen et al. / Molecular Phylogenetics and Evolution 23 (2002) 137–149

chondrial (mt) genome of Acropora tenuis indicated (a) that the organization of genes in that species distinctly differed from those in the nonscleractinian anthozoans, Metridium senile (Beagley et al., 1996; Pont-Kingdon et al., 1994) and Sarcophyton glaucum (Beaton et al., 1998; Pont-Kingdon et al., 1998), and (b) that transfer RNA (tRNA) import by mitochondria of Acropora tenuis is selective (Van Oppen et al., 1999b). Another unusual feature of scleractinian mt molecular evolution is that the evolutionary rate appears to be relatively slow, when genetic distances are compared to the divergence times of taxa based on fossil records (Chen and Yu, 2000; Romano and Palumbi, 1997) and geological events (Van Oppen et al., 1999a). Phylogenetic analyses using mt DNA sequences highlight the usefulness of a molecular approach for examining hypotheses of skeleton derivation and the phylogeny of scleractinian corals (Romano and Cairns, 2000; Romano and Palumbi, 1996, 1997). Analysis of a portion of the mitochondrial 16S ribosomal RNA (rRNA) gene (406–565 bp in PCR length) of 34 species of scleractinian corals, representing 29 genera in 14 of the 24 extant families, produced a quite different subordinal phylogeny from traditional phylogenies based on skeletal morphology (Romano and Palumbi, 1996, 1997). A subsequent study of mt 16S rRNA as well as a set of nuclear 28S ribosomal RNA markers in which the number of species examined was increased to 68 provides additional support for this phylogeny (Romano and Cairns, 2000). This mt-based phylogeny suggests that the Scleractinia are separated into two major clades named for the length of the 16S sequences: ‘‘short (robust)’’ and ‘‘long (complex)’’ clades, by Romano and Palumbi (1996). This contrasts with seven monophyletic groups which had been hypothesized from morphological-based phylogenies (Veron, 2000), Romano and Palumbi (1996) concluded that the separation of these two scleractinan clades, based upon molecular characters, occurred at least 300 mya. This separation would therefore have occurred before the first appearance of the scleractinian skeleton in the fossil record 240 mya, which implies that the scleractinian skeleton has evolved as many as four times from a soft-bodied, anemone-like ancestor. Although the two-clade phylogeny was strongly supported by relatively high bootstrap statistics under different molecular evolution analyses, lack of resolution within each of these two clades raised some questions. First, could slow-evolving mt 16S rRNA provide enough information to resolve the two clades if these lineages diverged from each other over a very short time period? Second, the divergence of the basal lineages was so long ago that the signal may have been lost from mt 16S rRNA (Romano and Cairns, 2000). These authors proposed that analyses of other gene regions were needed to provide better resolution within the ‘‘short’’ and ‘‘long’’ clades to delineate the relationships among basal

scleractinian lineages and among scleractinian families (Romano and Cairns, 2000). The other mitochondrial ribosomal gene, 12S rRNA, is a potential locus for examining the relationships among the basal scleractinian lineages and among scleractinian families. This gene has frequently been used to investigate familial- and ordinal-level phylogenies in several animal groups (Allard and Honeycutt, 1992; Hixson and Brown, 1986; Milinkovitch et al., 1993; Miyamoto et al., 1990). A ‘‘scleractinian-universal’’ primer set has been developed to amplify 80% of mt 12S rRNA from seven species of scleractinian corals, and surveys of sequence variation and estimation of the rate of evolution have shown an extremely slow divergence of this gene in the family Acroporidae (Chen and Yu, 2000). In addition, distinct size class differences of PCR products were observed among scleractinian corals, i.e., between five species of Acroporidae and two species of Faviidae corals (Chen and Yu, 2000). In this study, we used the mt 12S rRNA gene from 28 species of scleractinian corals to address the following questions: (1) Do the two distinct size classes of DNA sequences of mt 12S rRNA gene observed in the previous study (Chen and Yu, 2000) consistently appear in the other taxa, and, if so, do the size classes correspond to the two clades supported by the 16S rRNA gene phylogeny of scleractinian corals? (2) Does the slow rate of evolution of the mt 12S rRNA gene found in the family Acroporidae also occur in other coral families? (3) What is the level of phylogenetic resolution of mt 12S rRNA for scleractinan corals?

2. Materials and methods 2.1. Coral samples Taxa, sampling localities, and sources of DNA used in the present study are summarized in Table 1. This sampling included 28 species of scleractinian corals from 19 genera and eight families. Sperm were collected from Acropora muricata, A. digitifera, A. hyacinthus, A. cytherea, Mycedium elephantotus, Favia favus, Favites abdita, Galaxea astreata, and Platygyra sinensis from spawning colonies in southern Taiwan in 1997 (Dai et al., 1992) and in the Penghu Islands in 1998 and 1999 (Chen et al., unpubl. data) and were frozen in liquid nitrogen or dry ice for transfer to the laboratory. Branches of Acropora togianensis, A. palifera, A. brueggemanni, A. cuneata, A. digitifera, and A. muricata were collected from the Togian Islands, Sulawesi, Indonesia during the 1999 Tethyana expedition to Indonesia by the second and third authors. The remaining species, including the out group corallimorpharian, Rhodactis mussoides, were collected by the senior author from offshore islands around Taiwan. For these tissue

C.A. Chen et al. / Molecular Phylogenetics and Evolution 23 (2002) 137–149

139

Table 1 Taxonomic information, sampling localities, DNA source, GC content, and GenBank accession number of the mt SSU rRNA gene DNA source

PCR lengtha

Accession no.

Reference

Tissue

824

S75445

Pont-Kingdon et al. (1994)

Lanyu, Taiwan

Tissue

893

AF177049

Chen and Yu (2000)

Penghu Is., Taiwan Penghu Is., Taiwan

Tissue Tissue

704 702

AF333043 AF333044

Wanlitung, Taiwan

Tissue

874

AF177046

Montipora digitifera

Wanlitung, Taiwan

Tissue

874

AF177045

Montipora aequituberculata Anacropora sp.1 Acropora palifera

Penghu Is., Taiwan Togian Is., Sulawesi Togian Is., Sulawesi Penghu Is., Taiwan

Tissue Tissue Tissue Tissue

874 874 874 874

AF333045 AF333046 AF333047 AF177044

Togian Is., Sulawesi Togian Is., Sulawesi Togian Is., Sulawesi Togian Is., Sulawesi Wanlitung, Taiwan

Tissue Tissue Tissue Tissue Sperm

874 874 874 874 874

AF333048 AF333049 AF333050 AF333054 AF1177043

Togian Is., Sulawasi Penghu Is., Taiwan

Tissue Sperm

874 874

AF333052 API77042

Magnetic Is., Australia

Sperm

874

API52244

Penghu Is., Taiwan Nanwan Bay, Taiwan

Sperm Sperm

874 874

AF333053 AF333054

Wallace (1999) Wallace (1999)

Penghu Is,, Taiwan

Tissue

868

AF333055

Veron (1993)

Penghu Is,, Taiwan

Sperm

885

AF333056

Veron (1993)

Penghu Is., Taiwan

Sperm

694

AF333057

Veron (1993)

Penghu Is., Taiwan Penghu is., Taiwan

Tissue Tissue

693 690

AF333058 AF333059

Veron (1993) Veron (1993)

Penghu Is., Taiwan

Sperm

691

AF177048

Penghu Is., Taiwan Penghu Is., Taiwan

Sperm Sperm

691 692

AF333060 AF177047

Penghu Is., Taiwan Penghu Is., Taiwan Penghu Is., Taiwan

Tissue Tissue Tissue

693 692 703

AF333061 AF333062 AF333064

Veron (1993) Veron (1993) Veron (1993)

Penghu Is., Taiwan Penghu Is., Taiwan

Tissue Tissue

855 855

AF333063 AF333065

Veron (1993) Veron (1993)

Taxon

Sampling localities

Order Actiniaria Family Metridiidae Metridium senile Order Corallimorpharia Family Discosomidae Rhodactis mussoides Order Scleractinia Family Pocilloporidae Pocillopora damcornis Stylophora pistillata Family Acropolridae Astreopora myriophthalma

Acropora Acropora Acropora Acropora

brueggemanni cuneata togianesis digitifera

Acropora muricata

Acropora tenius Acropora hyacinthus Acropora cytherea Family Agariciidae Pavona frondifera Family Ocunlindae Galaxea astreata Family Penctiniidae Mycedium elephatotus Family Merulinidae Merulina ampliata Hydnophora exesa Family Faviidae Favia favus Favites abdita Platygyra sinensis Montastrea valenciennesi Cyphastrea chalcidium Oulastrea crispata Family Dendrophylliidae Turbinaria mesenterina Tubastrea aurea a

PCR length is in base pairs, and primers are excluded.

Identification

Chen unpublished data

Veron (1993) Veron (1993) Chen and Yu (2000) Chen and Yu (2000)

Veron (1993) Veron (1993) Veron (1993) Veron, 1993)

Chen and Yu (2000)

Wallace (1999) Wallace (1999) Wallace (1999) Wallace (1999)

Chen and Yu (2000)

Wallace (1999)

Chen and Yu (2000)

Wallace (1999)

Van Oppen et al. (1999b)

Wallace (1999)

Chen and Yu (2000) Chen and Yu (2000)

Veron (1993) Veron (1993) Veron (1993)

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samples, a small fragment of coral was clipped from each colony, placed in a labeled polyethylene bag, and preserved in 95% (v/w) ethanol. 2.2. DNA extraction, amplification, cloning, and sequencing DNA extraction was modified from methods described by Chen and Yu (2000) and Chen et al. (2000). Target segments of the mt 12S rRNA gene were amplified using the ‘‘scleractinian-universal’’ primer pairs, ANTMT12S-F: 50 -AGC CAC ACT TTC ACT GAA ACA AGG-30 and ANTMT12S-R: 50 -GTT CCC YYW CYC TYA CYA TGT TAC GAC-30 , described in Chen and Yu (2000). PCR was performed in a PC-9606 thermal sequencer (Corbett Research) using the following thermal cycle: 1 cycle at 95 °C (4 min); 4 cycles at 94 °C (30 s), 50 °C (1 min), 72 °C (2 min), and 30 cycles at 94 °C (30 s), 55 °C (1 min), and 72 °C (2 min). The amplification reaction used 50–200 ng of template and BRL Taq polymerase in a 50-ll volume reaction, using the buffer supplied with the enzyme, under conditions recommended by the manufacturer. The PCR products were electrophoresed in a 1% agarose (FMC Bioproduct) gel in 1 TAE buffer to assess the yield. Amplified DNA was extracted once with chloroform, precipitated with ethanol at )20 °C, and resuspended in TE buffer. PCR products were cloned using the pGEM-T system (Promega, Madison, WI) under conditions recommended by the manufacturer. Nucleotide sequences were determined for complementary strands of at least two clones from each sample using an ABI 377 Genetic Analyzer. The sequences obtained were submitted to GenBank under the accession numbers listed in Table 1. 2.3. Sequence alignment and phylogenetic analysis Complete DNA sequences of the mt 12S rRNA gene of Metridium senile (Pont-Kingdon et al., 1994) and Acropora tenius (Van Oppen et al., 1999b) retrieved from GenBank, and the secondary structure of Metridium senile (Pont-Kingdon et al., 1994) were used to facilitate alignment. DNA sequences were initially aligned using CLUSTAL W 1.7 (Thompson et al., 1994), followed by manual editing using SeqApp 1.9 (Gilbert, 1994), Sequence alignment within each clade was

straightforward, but numerous gaps were introduced in aligning the long and the short clades. The alignment indicated two groups with distinct PCR size ranges of mt 12S rRNA in scleractinian corals (Table 2). Variability in 30-bp sliding windows plotted throughout the aligned mt 12S rRNA gene was performed using MEGA 1.02 (Kumar et al., 1993). Aligned sequence data is available at the following website: www.sinica.edu.tw/ zool/english/cachen.htm/mt12S/12Salign.txt. Ambiguous regions were excluded from the phylogenetic analyses. Base composition bias was calculated using PAUP* 4.05 (Swofford, 2001), and a v2 test was performed to check for taxa with deviations of nucleotide composition. Significantly different nucleotide compositions between taxa may indicate the result of mitochondrial insertions with an evolutionary history different from that of the remaining taxa (Zhang and Hewitt, 1996). To investigate whether transitions and/or transversions in coral mt 12S rRNA genes may be saturated, pairwise substitution rates, defined as numbers of transitions or transversions divided by length of comparison, were plotted against uncorrected pairwise P distances (Li, 1997). Phylogenetic analyses were performed using PAUP* 4.05 (Swofford, 2001). Maximum-parsimony (MP) analyses were performed using heuristic searches with 100 random additions of sequences to search for the mostparsimonious trees. Bootstrapping with 1000 pseudoreplicates and a heuristic search were used to examine the robustness of clades in the resulting trees. Neighborjoining (NJ) analysis was performed based on TamuraNei distances (Tamura and Nei, 1993), correcting for unequal base frequencies and different transition and transversion rates, The robustness of NJ phylogenies was assessed by the 1000 bootstrap option, For maximum-likelihood (ML) analysis, the best-fit model of DNA substitution and parameter estimates used for tree construction were chosen by performing hierarchical likelihood ratio tests (reviewed in Harris and Crandall, 2000; Huelsenbeck and Crandall, 1997) using PAUP 4.05 (Swofford, 2001) and Modeltest 3.0 (Posada and Crandall, 1998). Likelihood-ratio tests indicated that the Tamura-Nei model with the general-time-reversible option was the most appropriate for subsequent ML analysis (Table 4). Heuristic ML searches were performed using fast stepwise-addition and 500 bootstrap

Table 2 Base composition, A–T bias, substitutions, distance, and probabilities of the v2 test of nucleotide bias among all sequences

Long Short Long + Short a

A

T

C

G

A–T biasa

v2

df

P

Ts/Tvb

Distancec

0.3208 0.3404 0.3288

0.2973 0.3101 0.2966

0.1473 0.134 0.1419

0.2445 0.2155 0.2327

61.81 65.51 62.54

2.508 1.865 37.001

45 30 78

1.0000 1.0000 0.999

2.03 1.948 1.37

2.18 7.16 12.85

All values of base composition are in percent (%). Mean among pairwise comparison. c Uncorrected P distance. b

C.A. Chen et al. / Molecular Phylogenetics and Evolution 23 (2002) 137–149

pseudoreplicates. Tree comparisons between MP and ML analyses were tested using the constraint option in PAUP4.05 as well as Templeton (Templeton, 1983) and Kishino–Hasegawa (Kishino and Hasegawa, 1989) tests as implemented in PAUP 4.0b4. In addition, a molecular clock likelihood ratio test (LRT), 2D ¼ log Lno clock  LogLclock , which is distributed as v2 with (n  2) degrees of freedom, where n is the number of sequences (Muse and Weir, 1992), was also performed by TREE-PUZZLE 5.0 (Schmidt et al., 2000) to determine whether there was a statistical difference in evolutionary rates between the long and short clades.

141

different families in the long clade (e.g., Acroporidae vs Dendrophyllidae) (Fig. 2A). The within-family variability in the short clade is low except for the comparisons of Oulastrea crispata and other Faviidae corals. Variability was also low in the comparisons among species of Faviidae other than Oulastrea, Mycedium elephantotus, Merulina ampliata, and Hydnophora exesa, but was higher when comparisons were conducted between Pocilloporidae and Faviidae corals (Fig. 2B). Comparisons between the two clades showed extremely high variability at several poorly aligned regions spanning the sequence (Fig. 2C). 3.2. Phylogenetic analyses

3. Results 3.1. Sequence characteristics of the mt 12S rRNA gene in corals Sequencing of the mt 12S rRNA PCR fragments, resulted in two distinct clades (Fig. 3): one which we refer to as the ‘‘long’’ clade, had PCR product lengths ranging from 855 base pairs (bp) in Tubastrea aurea and Turbinaria mesenterina to 885 bp in Galaxea astreata; and the second, which we refer to as the ‘‘short’’ clade, had PCR product lengths ranging from 690 bp in Hydnophora exesa to 704 bp in Stylophora pistillata (Table 1). Both long and short clades showed nucleotide composition bias toward A and T with a mean composition of 61.81% in the long clade and 65.51% in the short clade. However, base composition did not differ significantly between taxa when the two clades were considered separately or as a single group (Table 2, v2 test, P > 0:999). In the long clade, the transition vs transversion (Ts/Tv) ratios ranged from 0 to 3.167 with a mean of 2.03 (Table 2). No transversion substitutions were observed in several pairwise comparisons between species in the family Acroporidae (Table 3). In the short clade, the Ts/Tv ratio was substantially more variable, ranging from 0 to 8 with a mean of 1.95 (Table 2). Within-family pairwise comparisons showed very high Ts/Tv ratios, including Favites abdita vs Cyphastrea chalcidium (8), F. abdita vs Hydnophora exesa (6), C. chalcidium vs Montastrea valenciennesi (6), C. chalcidium vs Mycedium elephantotus (7), C. chalcidium vs Merulina ampliata (8) (Table 3). The overall mean of Ts/Tv including both clades was 1.37 (Table 2). Overall, transitions accumulated more quickly than transversions in both clades (Fig. 1). The mean P distance was 2.18% for the long clade, and 7.16% for the short clade. Between the two ctades the mean P distance was 12.85% (Tables 2 and 3). Spatial patterns of nucleotide substitution in 30-bp window comparisons indicated that variability was extremely low between species within the genus Acropora and among different genera within the family Acroporidae (data not shown). It is also relatively low between

The outgroup sequences used in the phylogenetic analyses were the mt 12S rRNA sequences of the actiniarian, Metridium senile, and the corallimorpharian, Rhodactis mussoides. Sequences of 924 bp for each species were considered for alignment. Ambiguous regions of poor alignment as indicated in Fig. 2C were eliminated after manually adjusting the alignment of secondary structures of Metridium senile (Pont-Kingdon et al., 1994), and 643 bp of the final alignment was subsequently used for phylogenetic analyses. Of the 643 characters in the alignment, 289 (44.9%) were variable and 192 (29.9%) were parsimony informative. In order to decrease computing time for phylogenetic inferences, 11 species in the family Acroporidae were included. Replacing species in repeated analyses did not change the overall topology or arrangement of species in the Acroporidae branch, presumably due to the similarity of mt 12S rRNA sequences in this family. Parsimony analysis revealed a single MP tree with tree length of 519, a consistency index of 0.741, a retention index of 0.896, and a rescaled consistency index of 0.644. Neighborjoining (NJ) analysis produced a NJ tree identical to that of MP topology (Fig. 3A). Both MP and NJ trees supported the two clade grouping with high bootstrap values separating the clades in both analyses (Fig. 3A). In the long clade, the family Acroporidae formed a monophyletic group with a short tree length. The azooxanthellate coral, Tubastrea aurea clustered with its confamilial zooxanthellate species, Turbinaria mesenterina, as supported by 100% bootstrap. Taxa within the short clade fell into two groups also with 100% bootstrap support. One group consists of two genera in the family of Pocilloporidae, and the other includes species from the families Pectiniidae, Merulinidae, and Faviidae. Within the family Faviidae, Oulastrea crispata showed the highest divergence, branching from the other Faviidae species with 85% bootstrap support in NJ analysis, but not in MP analysis (data not shown). Based on the results of the Modeltest likelihood ratio test (Table 4), the ML analysis of mt 12S rRNA yielded the topology shown in Fig. 3B ( ln likeli-

1.566 1.727 1.814 1.814 1.791 1.791 1.814 1.791 1.767 1.814 1.773 1.791 1.791 1.408 1.447 1.540 1.733 1.080 1.103 1.119 1.095 1.084 1.098 1.096 0.922 1.107 1.095 1.096

METR RHDA ASTRa ANACa MONAa MONDa APALa ABREa ACUTa ATOGa ADIGa AMURa ATENa AHYAa TUBAa TURBa GALAa PAVOa POCIb STYLb FAVIb FAVTb CYPHb MONTb PLATb OLASb MYCEb MERUb HYDOb

2.444 2.577 2.538 2.538 2.538 2.577 2.538 2.577 2.538 2.481 2.538 2.538 2.143 2.308 2.065 2.214 1.127 1.203 1.027 1.013 1.041 1.027 1.056 1.026 1.027 1.040 1.027

0.2124

RHDA

2 1 0 0 1 0 1 1 1 0 0 2.222 2.714 1.833 1.818 0.971 1.015 0.909 0.879 0.892 0.875 0.905 0.750 0.894 0.879 0.877

0.1866 0.1452

ASTRa

–c – – – – – – 2 – – 2.500 3.167 1.909 2 1.015 1.060 0.938 0.908 0.922 0.905 0.935 0.789 0.923 0.908 0.906

0.1882 0.145 0.0047

ANACa

– – – – – – 1 – – 2.375 3 1.909 1.9 1 1.045 0.923 0.892 0.906 0.889 0.919 0.775 0.908 0.892 0.891

0.1882 0.1435 0.0031 0.0016

– – – – – 2 – – 2.500 3.167 2 2 1 1.045 0.938 0.908 0.922 0.905 0.935 0.775 0.923 0.908 0.906

0.1866 0.1437 0.0016 0.0031 0.0016

– – – – 2 – – 2.500 3.167 2 2 1 1.045 0.938 0.908 0.922 0.905 0.935 0.775 0.923 0.908 0.906

0.1866 0.1437 0.0016 0.0031 0.0016 0

MONAa MONDa APALa

– – – 3 – – 2.625 3.333 2.091 2.1 0.985 1.03 0.923 0.892 0.906 0.889 0.919 0.761 0.908 0.892 0.891

0.1882 0.1452 0.0031 0.0047 0.0031 0.0016 0.0016

ABREa

– – 2 – – 2.500 3.167 2 2 1 1.045 0.938 0.908 0.922 0.905 0.935 0.775 0.923 0.908 0.906

0.1866 0.1437 0.0016 0.0031 0.0016 0 0 0.0016

ACUTa 0.1882 0.1435 0.0031 0.0016 0 0.0016 0.0016 0.0031 0.0016 0.0031 1 – – 2.375 3.000 1.909 1.9 1 1.045 0.923 0.892 0.906 0.889 0.919 0.775 0.908 0.892 0.891

3 – – 2.625 3.333 2.091 2.1 1 1.060 0.954 0.923 0.938 0.921 0.952 0.789 0.938 0.923 0.922

ADIGa

0.1851 0.1452 0.0031 0.0047 0.0031 0.0016 0.0016 0.0031 0.0016

ATOGa

2 2 2.222 2.714 1.833 1.818 1 1.044 0.894 0.864 0.877 0.859 0.839 0.778 0.879 0.864 0.862

0.1897 0.1466 0.0062 0.0047 0.0031 0.0047 0.0047 0.0062 0.0047 0.0062 0.0031

– 2.500 3.167 2 2 1 1.045 0.938 0.908 0.922 0.905 0.935 0.775 0.923 0.908 0.906

0.1866 0.1437 0.0016 0.0031 0.0016 0 0 0.0016 0 0.0016 0.0016 0.0047

AMURa ATENa

2.500 3.167 2 2 1 1.045 0.938 0.908 0.922 0.905 0.935 0.775 0.923 0.908 0.906

0.1866 0.1437 0.0016 0.0031 0.0016 0 0 0.0016 0 0.0016 0.0016 0.0047 0

0.5 2.545 1.833 1.045 1 0.821 0.821 0.833 0.785 0.813 0.812 0.806 0.791 0.788

0.1837 0.1374 0.0451 0.0436 0.042 0.0436 0.0436 0.0451 0.0436 0.0451 0.042 0.0451 0.0436 0.0436

AHYAa TUBAa

3 2.2 1.109 1.058 0.836 0.836 0.848 0.800 0.828 0.812 0.821 0.806 0.803

0.1792 0.1343 0.0405 0.039 0.0374 0.039 0.039 0.0405 0.039 0.0405 0.0374 0.0405 0.039 0.039 0.0047

TURBa

2.545 1.123 1.059 0.909 0.894 0.923 0.906 0.937 0.806 0.909 0.894 0.892

0.1975 0.1481 0.0529 0.0498 0.0498 0.0513 0.0513 0.0529 0.0513 0.0529 0.0498 0.0529 0.0513 0.0513 0.0607 0.0561

GALAa

1.175 1.141 1.000 0.952 0.984 0.967 0.952 0.836 0.968 0.952 0.968

0.1913 0.1403 0.0482 0.0467 0.0451 0.0467 0.0467 0.0482 0.0467 0.0482 0.0451 0.0482 0.0467 0.0467 0.0529 0.0498 0.0607

PAVOa

1.538 0.98 1.021 1 1 0.959 0.891 1.021 1.021 0.959

0.2878 0.2643 0.2139 0.2153 0.2137 0.2139 0.2139 0.2123 0.2139 0.2139 0.2137 0.2168 0.2139 0.2139 0.2126 0.2128 0.2168 0.2154

POClb

1.205 1.209 1.238 1.244 1.167 1.04 1.209 1.209 1.190

0.2877 0.2565 0.2154 0.2169 0.2153 0.2154 0.2154 0.2138 0.2154 0.217 0.2153 0.2184 0.2154 0.2154 0.2236 0.2237 0.22 0.2154 0.0519

STYLb

1.8 2 1.333 0.75 1.219 1.2 1.4 0.75

0.2771 0.2374 0.1962 0.1962 0.1946 0.1962 0.1962 0.1946 0.1962 0.1977 0.1946 0.1946 0.1962 0.1962 0.1903 0.1919 0.1962 0.1962 0.1591 0.1527

FAVlb

8 3.5 2 1.4 – – 6

0.2737 0.2355 0.1928 0.1928 0.1913 0.1928 0.1928 0.1913 0.1928 0.1944 0.1913 0.1913 0.1928 0.1928 0.19 0.1916 0.1944 0.1913 0.1525 0.1494 0.0218

FAVTb

6 2.5 1.448 7 8

0.2691 0.2355 0.1913 0.1913 0.1897 0.1913 0.1913 0.1897 0.1913 0.1928 0.1897 0.1897 0.1913 0.1913 0.1885 0.1901 0.1944 0.1913 0.1541 0.1478 0.0187 0.014

CYPHb

1 1.167 2 2.5 1

0.2675 0.2308 0.1866 0.1866 0.1851 0.1866 0.1866 0.1851 0.1866 0.1882 0.1851 0.1851 0.1866 0.1866 0.1807 0.1823 0.1897 0.1866 0.151 0.1447 0.0109 0.014 0.0109

1.161 1 1.333 0

0.2706 0.2300 0.1866 0.1866 0.1851 0.1866 0.1866 0.1851 0.1866 0.1882 0.1851 0.1851 0.1866 0.1866 0.1807 0.1823 0.1897 0.1882 0.151 0.1431 0.0109 0.014 0.0109 0.0031

MONTb PLATb

1.3 1.267 1.241

0.2699 0.2412 0.1967 0.1983 0.1967 0.1967 0.1967 0.1951 0.1967 0.1983 0.1967 0.1998 0.1967 0.1967 0.1955 0.1955 0.203 0.192 0.1643 0.161 0.1109 0.1124 0.1109 0.1015 0.1046

OLASb

– 3

0.2753 0.237 0.1944 0.1944 0.1928 0.1944 0.1944 0.1928 0.1944 0.196 0.1928 0.1928 0.1944 0.1944 0.1885 0.1901 0.196 0.1928 0.1525 0.1494 0.0171 0.0078 0.0124 0.0093 0.0093 0.1077

4

0.2737 0.2386 0.1928 0.1928 0.1913 0.1928 0.1928 0.1913 0.1928 0.1944 0.1913 0.1913 0.1928 0.1928 0.1869 0.1885 0.1944 0.1913 0.1525 0.1494 0.0187 0.0093 0.014 0.0109 0.0109 0.1062 0.0016

0.2709 0.2342 0.19 0.19 0.1884 0.19 0.19 0.1884 0.19 0.1915 0.1884 0.1884 0.19 0.19 0.184 0.1857 0.1917 0.19 0.1512 0.1448 0.0109 0.0109 0.0093 0.0031 0.0031 0.1017 0.0062 0.0078

MYCEb MERUb HYDOb

Note. Taxon abbreviations: METR: Metridium senile; RHDA: Rhodactis mussoides’; ASTR: Astreopora myriophthalma; ANAC: Anacropora sp1; MONA: Montipora aequituberculata; MONO: Montipora digitata; APAL: Acropora palifera; ABRE: Acropora brueggemanrti; ACUT: Acropora cuneata; ATOG: Acropora togianesis. ADIG: Acropora digitifera; AMUR: Acropora muricata; ATEN: Acropora tenuis, AHYA: Acropora hyadnthus, TUBA: Tubastrea aurea; TURB: Turbinaria mesenterina; GALA: Galaxea astreata; PAVO: Pavona frondifera; POCI: Pocillopora damicomis; STYL: Stylophora pistillate; FAVI: Favia favus; FAVT: Favites abdita; CYPH: Cyphastrea chalcidium, MONT: Montasrea valenciennesi; PLAT: Platygyra sinensis; OLAS: Oulastrea crispata; MYCE: Mycedium elephantotus; MERU: Merulina ampliata; HYDO: Hydnophora exesa. a Taxon belonging to the long clade as indicated in Fig. 3. b Taxon belonging to the short clade as indicated in Fig. 3. c Transition/transversion ratio is not available due to no transversion occurring in that taxon.

METR

Taxon

Table 3 Pairwise P distance (upper triangular matrix) and transition/transversion ratio (lower triangular matrix) for the range of taxa used

142 C.A. Chen et al. / Molecular Phylogenetics and Evolution 23 (2002) 137–149

C.A. Chen et al. / Molecular Phylogenetics and Evolution 23 (2002) 137–149

143

Fig. 1. Relationship between genetic distance and transition and transversion rates for possible pairwise comparisons of (A) long sequences (855–885 bp) and (B) short sequences (690–704 bp).

hood ¼ 3345.17). The ML tree formed an unsolved polytomy with low bootstrap support (69%) at the basal lineage. Species from the family Acroporidae did not form a monophyletic ‘‘long’’ clade with Turbinaria mesenterina, Tubastrea aurea, Galaxea astreata, and Pavona frondifera as indicated in the MP and NJ analyses (Fig. 3A). The short clade did, however, form a monophyletic group with 100% bootstrap support. The ML tree significantly differed from the MP tree by Kishino– Hasegawa and Templeton Wicoxon signed-ranking tests, and the tree length of the MP tree was 15 steps shorter than that of the ML tree (Table 5). The molecular-clock hypothesis tested by LRT was rejected for mt 12S rRNA ( log Lno clock ¼ 3223:24,  log Lclock ¼ 33339:96, df ¼ 27, P < 0:000001), suggesting that the evolutionary rate of these two clades of mt 12S rRNA gene was not constant over time in any of the scleractinian lineages (Table 4).

4. Discussion 4.1. Mitochondrial 12S rRNA gene in scleractinian corals

Fig. 2. Variability in 30-bp sliding windows plotted throughout the mt 12S rRNA gene: (A) two long sequences, Acropora digitifera vs Tubastrea aurea; (B) two short sequences, Stylophora pistiilata vs Platygyra sinensis; and (C) a long and a short sequence, Acropora tenuis vs Oulastrea crispata. Poorly aligned regions in the alignment of long and short sequence were indicated by stars.

The amplified PCR products of the scleractinian mitochondrial 12S rRNA gene formed two major clades, a ‘‘short clade’’ in which sequences ranged from 690 to 704 bp in length and a ‘‘long clade’’ with sequences of 855 to 885 bp in length. Several molecular characteristics reported for the scleractinian mt 16S rRNA gene (Romano and Palumbi, 1997) were also observed in the present study in the 12S rRNA gene despite the use of different exemplar species. First, nucleotide composition of the sequences from both major clades showed a slight bias toward A and T, and did not differ significantly from each other, Second, the grouping of scleractinian genera into these two major clades corresponds to those

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Fig. 3. Phylogenetic analyses of the mt 12S rRNA gene. (A) Relationships based on MP and NJ algorithms. The most-parsimonious tree found using the heuristic search option with 100 replicates of random-addition sequences with 1000 bootstraps and NJ tree found using Tamura–Nei distance with 1000 bootstraps. Bootstrap values > 50% are given above and below the corresponding branches; the value above the branch is the parsimony analysis bootstrap percentage, and below is from NJ analysis, (B) ML tree of scleractinian corals based on 12S rRNA sequences. The topology was reconstructed under the TrN + G model of nucleotide substitution, Boostrap estimates are based on 100 replicates, Bootstrap values < 50% are not shown.

in the phylogeny derived from the mt 16S rRNA gene (Romano and Cairns, 2000; Romano and Palumbi, 1996, 1997), This correlation between phylogenetic pathways of the 12S and 16S rRNA genes may reflect the fact that both genes are linked on the mitochondrial genome, and are thus inherited as a single locus, or that the functional constraints of both ribosomal genes which integrate into functional ribosomes (Noller, 1984) are involved in protein synthesis (reviewed in Dahlberg, 1989; Gerbi, 1985). Further sampling of 12S rRNA gene sequences from exemplar species are required to examine this correlation. Third, pairwise comparisons of the average Ts/Tv ratio of the 12S rRNA gene from the present study and those of 16S rRNA gene (Table 2 in Romano and Palumbi, 1997) all show that transitional substitutions occur more frequently than transversions. A bias in the transition vs transversion ratio is often reported, with transitions usually more frequent than transversions, particularly in mitochondrial sequence comparisons (DeSalle et al., 1987). However, the pro-

portion of transitions found between diverging DNA sequences is known to decrease with increasing divergence time (Brown et al., 1982; DeSalle et al., 1987; Mindell and Honeycutt, 1990). Characteristically the Ts/Tv ratio of recently diverged sequences is usually 2.0 or more. In comparisons between the long and short clades, Ts/Tv ratios of 12S rRNA gene decline to 1.0 or lower. In contrast, Ts/Tv ratios between genera within each clade reach 2.0 or higher (Table 3), indicating that divergence within each clade is not ancient. The spatial distribution of nucleotide substitutions shows different patterns between the ‘‘long’’ and ‘‘short’’ clades (Figs. 2A and B), despite the fact that the two clades share similar AT contents and average transition/ transversion ratios, In 30-bp window comparisons of 12S rRNA sequences, the long clade showed low variability along the sequences while the short clade showed much higher variability at both the 50 - and 30 -ends of sequences, indicating that sequences of the short clades

C.A. Chen et al. / Molecular Phylogenetics and Evolution 23 (2002) 137–149

145

Fig. 3. (continued).

possess higher within-clade variation (7.16% of P distance) than those of the long clade (2.18%). These comparisons provide evidence that the evolutionary rate of 12S rRNA sequences is not equal between the two clades. The nucleotide substitution patterns of the 16S rRNA gene are similar in both clades (Romano and Palumbi, 1997). 4.2. Evolutionary rate of the mt 12S rRNA gene in scleractinian corals The evolutionary rates of molecules are usually estimated by comparing sequences from the same gene region for different taxa with a similar first date of appearance in the fossil record (Li, 1997). Therefore, accurate estimates of evolutionary rates depend upon well-preserved fossil records and a constant rate of di-

vergence of molecules in different evolutionary lineages (molecular clock hypothesis). Fossil records of genera and families have been documented in scleractinian corals (Wells, 1956; reviewed in Veron, 1995, 2000), enabling the divergence rate of the mt 12S rRNA gene to be estimated for this group. Several patterns of divergence rates of mitochondrial ribosomal genes in scleractinian corals can be discerned. First, in species of Scleractinia, the 12S and 16S rRNA genes evolved at different rates, whereas divergence rates of both these genes in Drosophilia are similar (Table 6). This is probably due to different evolutionary constraints acting in corals and in Drosophilia. Second, a relatively slower rate of evolution of the 12S rRNA gene (Chen and Yu, 2000) appears to be a common feature of taxa within the long clades, but is not observed in the short clade. For example there was a similarly low rate of divergence

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Table 4 Test of hypotheses relating to models of evolution appropriate for phylogeny reconstruction (Huelsenbeck and Crandall, 1997) and molecular clock ratio test (Muse and Weir, 1992) for examining whether a molecular clock exists among species Null hypothesis

Model compared a

b

H0 : JC69 H1 : F81 H0 : F81 : H1 : HKY85c H0 : HKY85; H1 : TrNd H0 : TrN; H1 ; TIMe H0 : TrN; H1 : TrN þ Gf H0 : TrN þ G; H1 : HKY85 þ G þ invarg H0 : without clock; H1 : with clock

Equal base frequencies Equal transition/transversion rate Equal transition rates Equal transversion rates Equal rate among sites No invariable sites Molecular clock likelihood ration test

 ln L0

 ln L1

df

P

3458.0527 3419.0945 3341.1394 3336.8848 3336.8848 3249.9995 3223.24

3419.0945 3341.1394 3336.8848 3336.0703 3249.9995 3249.4910 3339.96

3 1 1 1 1 1 27

< 0:00001 < 0:00001 < 0:00353 < 0:20185 < 0:00001 < 0:31321 < 0:00001

a

JC69, Jukes and Cantor (1969). F81, Felsentstein (1981). c HKY85, Hasegawa et al. (1985). d TrN, Tamura and Nei (1993). e TIM, Posada and Crandall (1998). f G, gernal time reversible, Rodriguez et al. (1990). g invar, proportion of invariable sites. b

Table 5 Testing topology congruence of the maximum-pasimony (MP) and maximum-likelihood (ML) trees Topology

MP ML

MP ML

Tree length

519 534

519 534

Length difference

15

Kishino–Hasegawa test SD

t

P

5.357

2.8

0.0053

Templeton (Wilcoxon signed-ranks)

Winning-sites tests

Rank sum

n

z

P

Count

P

98

14

3:153

0.0016

13

0.0018

Note. t, Statistic of Kishino–Hasegawa test. P, probability of Kishino–Hasegawa test/Templeton test of finding a topology that does not differ from that of the MP tree; n, number of most-parisimonious trees; z, statistic of Templeton (Wilcoxon signed-ranks) test. ** Significant at P < 0:01.

Table 6 Divergence rates calculated from mitochondrial ribosomal RNA genes Taxa compared

Divergence rate (%/mya) Scleractiniana

Genera Family

Taxa compared b

12S (long clade)

12S (short clade)

16S

0–0.0088 0.037–0.072

0.004–0.277 0.013–0.164

0.02–0.04 0.06–0.12

Species Family

Drosophila 12Sc

16Sb

0.066–0.16c 0.078–0.086d

0.05–0.2 0.07

a

Estimation of divergence times of scleractinian fossil records refer to Veron (1995) and Wells (1956). Data were adopted from Romano and Palumbi (1997). c Drosophila divergences were compared from 665–688 bp of a segment of 12S sequences from D. bifasciata, D. melanogaster, D. subsilvestris, D. willistoni (Simon et al., 1996); the divergence data was set to 60 mya. d Comparisons were done between a 268-bp portion of 12S sequence of Aedes albopictus (Shouche and Patole, 2000; GenBank accession no. AF034471) and the homologous region of Drosophila spp.; the divergence date was set to 200 mya. b

(0.0047%/mya) between the azooxanthellate coral Tubastrea aurea and its zooxanthellate counterpart, Turbinaria mesenterina, both from the family Dendrophyllia. The divergence rate of the long clade is comparatively slower than that of Drosophilla at the interspecific level (Table 6), which has similar divergence dates to scleractinian genera (40–60 mya; Wells, 1956). In the short clade, the intergeneric divergence rate of mt

12S rRNA sequences varied from 0.004 to 0.277%/mya, indicating that the divergence rates among different genera in the short clade are not as homogeneous as those of the long clade. This probably reflects the early split between the family Pocilloporidae and other robust corals in the short clade, and among Oulastrea crispata and other Faviidae corals (Fig. 3). Third, the patterns of 12S divergence rates differ between the two major clades:

C.A. Chen et al. / Molecular Phylogenetics and Evolution 23 (2002) 137–149

the long clade diverged at a slower rate than the short clade (Table 6). This difference is consistent with results from the likelihood ratio test of the phylogenetic tree (Fig. 3). With one exception, this difference was not reported in scleractinian 16S rRNA (Romano and Cairns, 2000). These implications, however, should be considered conservatively, because relative rate difference could be biased due to the unequal sampling of taxa in each clade (Robinson et al., 1998). In the ‘‘complex corals,’’ 13 of the 17 species analyzed come from the family Acroporidae, and nine of those come from the genus Acropora. For the ‘‘robust corals’’ only 11 species, with six from family Faviidae were sampled. Although the results are supported by statistic test, further sampling of other congeneric and confamilial species in the short clade is necessary to reexamine this conclusion. The evidence presented in this study demonstrates that all of the scleractinian sequences are from the mt 12S rRNA gene, and that there was an early split in the ancient coral that led to that major clades of corals with short or long sequences. However, as discussed by Romano and Palumbi (1997) the split of coral families between long and short clades stands in strong contrast to traditional views of coral taxonomy at the subordinal level (Veron, 1995, 2000). Therefore, alternative hypotheses about the evolution of short and long clades of mt 12S rRNA gene should be considered. These alternative hypotheses include the possible contamination of PCR reactions, derivation from a mitochondrial pseudogene in the nuclear genome (Lopez et al., 1994; Zhang and Hewitt, 1996), duplication of the mt 12S rRNA gene within the mitochondrial genome (Moritz and Brown, 1986, 1987), and existence of gender-specific mitochondrial genomes (Zouros et al., 1994). The likelihood of these alternative hypotheses has been extensively reviewed by Romano and Palumbi (1997) who rejected this as an explanation of the persistence of the two distinct clades of mitochondrial 16S rRNA gene during the scleractinian evolution. The best-supported hypothesis is that both the short and long sequences of 16S rRNA are from the mt gene region and that both are functional, and that divergence between two clades represents divergence of two lineages of coral species not the duplication of two loci (Romano and Palumbi, 1997). The same scenario is applicable to the mt 12S rRNA gene, because the 12S and 16S rRNA genes are linked together on the mitochondrial genome, and the grouping of scleractinian genera into these two major clades is supported in the phylogenies constructed from both genes. 4.3. Phylogeny of the scleractinia based on the mt 12S rRNA gene Phylogenetic congruences and conflicts between molecular, morphological, and fossil data for the sclerac-

147

tinian suborder and relationships between families are discussed in Romano and Cairns (2000). Using the 16S rRNA gene, they found robust evidence for two major clades and estimated the date of their divergence to be as early as 300 mya, suggesting that the scleractinian skeleton could have evolved as many as four times from a soft-bodied anemone or corallimorpharian-like ancestor (Romano and Cairns, 2000). Although the basal lineages form an unresolved polytomy in the 12S rRNA ML tree, the MP (NJ) topology is significantly shorter than that of the ML tree (Fig. 3). The two-clade hypothesis is further supported by high bootstrap statistics. Within-clade relationships are also highly similar to those of the 16S phylogeny, although our sampling size across families and genera was small in comparison to the 16S data set (Romano and Cairns, 2000; Romano and Palumbi, 1996, 1997). For example, neither the 12S nor the 16S data support a monophyletic origin for Pocilloporidae and Acroporidae (both currently in the suborder Archaeocoeniia), as each of them is grouped with other families in different clades. This conflicts with the contention that modern coral taxa appear to be broadly divisible into two groups: the Archaeocoeniia and all other suborders, including Fungiina, Faviina, and Caryophyllina (Veron, 1995, 2000). In the short clade, most families of the suborder Faviina form a monophyletic group, except that the Faviidae genus, Oulastrea, diverges early from the group (Fig. 3). Oulastrea crispata is an unusual species with an encrusting or massive growth form which tolerates extremely low temperatures (Yajuma et al., 1986) and turbid conditions and occurs well into the temperate zone (Honma and Kitami, 1978). Whether these unusual features can account for the apparent early divergence of O. crispata from other Faviidae genera needs further examination of generic phylogeny of the family Faviidae and other families of the suborder Faviina. Nevertheless, phylogenetic resolution beyond the family level using either mt 12S rRNA gene of 16S rRNA gene (Romano and Palumbi, 1997) is limited. This constraint is probably due to the slow evolution of the scleractinian mitochondrial genome. Other faster-evolving nuclear loci, including complete DNA sequences of 28S rRNA encoding gene (Chen et al., 2000) and protein-coding genes (Chen et al., unpublished data), are currently under investigation to resolve these issues. In conclusion, analysis of a portion of the mt 12S ribosomal gene from 28 species of scleractinian corals suggests that the evolutionary rate of 12S is unequal among the lineages. Phylogenetic analyses of this region do not support the current view of scleractinian relationships based on skeletal morphology and fossil records. On the contrary, 12S rRNA phylogeny indicates that the scleractinian skeleton could have evolved at least twice. The 12S rRNA phylogeny also supports the

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two-clade hypothesis derived from the mt 16S ribosomal gene (Romano and Cairns, 2000) that indicates the divergence of the two clades occurred at least 300 mya.

Acknowledgments Many thanks to the staff of the Penghu Aquarium, a facility of the Taiwan Fishery Research Institute, for providing assistance during coral spawning in 1998 and 1999. We thank B. Rosen, C.-F. Dai, P. Muir, and members of the Evolution and Ecology Discussion Group. Institute of Zoology, Academia Sinica (IZAS), and two anonymous reviewers for constructive comments. This work was supported by grants from IZAS, the National Science Council, Republic of China (CAC), and the Australian Research Council (CCW). JW is the receipt of the Lizard Island Fellowship. This is Evolution and Ecology Group, IZAS Contribution no. 11.

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