Zoosyst. Evol. 99 (1) 2023, 135-147 | DOI 10.3897/zse.99.96386 Atte BERLIN Species delimitation, molecular phylogeny and historical biogeography of the sweetlips fish (Perciformes, Haemulidae) Ehsan Damadi', Faezeh Yazdani Moghaddam!*, Mehdi Ghanbarifardi® 1 Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran 2 Zoological Innovations Research Department, Institute of Applied Zoology, Faculty of Science, kerdowsi University of Mashhad, Mashhad, Iran 3 Department of Biology, Faculty of Science, University of Sistan and Baluchestan, Zahedan, Iran https://zoobank. org/83B2A 176-7001-4EDD-BF'53-DEC6EE24E582 Corresponding author: Faezeh Yazdani Moghaddam (yazdani@um.ac. ir) Academic editor: Nalani Schnell # Received 20 October 2022 Accepted 3 February 2023 Published 23 February 2023 Abstract The subfamily Plectorhinchinae (sweetlips) is composed of poorly-known species with high commercially and ecologically val- ues that exhibit phenotypic plasticity and various morphologies. Few studies have assessed the validity of sweetlips, intergeneric relationships and evolutionary survey in this subfamily, which have not yet been resolved. This study investigated the DNA se- quences of (1) the mitochondrial COI gene to delimit species, and (2) two mitochondrial (COI and Cyt b), and one nuclear (RAG1) markers to infer phylogenetic relationships and evolutionary and biogeographic history. The molecular results could differentiate Diagramma punctatum from the other species, but failed to distinguish D. /abiosum as a distinct species with considerably lower genetic distances for the COI (0.53%) and Cyt b (0.51%) markers. However, additional taxonomic investigations are required to shed light on this issue. All previously described nominal species of sweetlips in the northwest Indian Ocean were found to be well supported. The monophyly of Plectorhinchus is not supported and Diagramma pictum and D. punctatum should be assigned to the genus Plectorhinchus. The biogeographic history of Plectorhinchinae likely originated in the Indo-Pacific ca. 34 Ma (30-39 Ma; late Eocene/ middle Oligocene) and subsequently colonised the Western Indian Ocean and the Central Indo-Pacific. Maximum di- versification within the subfamily occurred from the middle Miocene to Pliocene, coinciding with dispersal and vicariance events. Diversification was probably driven by both biological and geographical factors. Key Words biogeography, mito-nuclear, molecular systematics, Plectorhinchus Introduction 21 genera and 136 species (Fricke et al. 2022). The Plec- torhinchinae commonly known as sweetlips, inhabit the Accurate species delimitation and phylogenetic recon- — shallow coastal waters of the Indo-West Pacific and East- struction are vital to understand biodiversity assessments, conservation management, evolutionary patterns, and processes (Traldi et al. 2020; McCord et al. 2021). Mem- bers of the family Haemulidae Gill, 1885 are dominant in shallow coral or rocky reefs of tropical and subtropical waters worldwide (McKay 2001; Lindeman and Toxey 2003; Nelson et al. 2016). They also have commercial and ecological values for most nations (Rocha et al. 2008). The family comprises two subfamilies Haemulinae Gill, 1885 and Plectorhinchinae Jordan & Thompson, 1912, ern Atlantic, which currently comprises 42 recognised species (Fricke et al. 2022). The species are classified into four genera (Diagramma Oken, 1817, Genyatremus Gill, 1862, Parapristipoma Bleeker, 1873 and Plectorhinchus Lacépede, 1801), of which Plectorhinchus is the most speciose (Fricke et al. 2022); however, previous molecu- lar studies have shown that Genyatremus does not belong to the Plectorhinchinae (Bernardi et al. 2008; Sanciangco et al. 2011; Tavera et al. 2018). Ever since the validation of Diagramma and Plectorhinchus, the identification of Copyright Damadi, E et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 136 haemulid species, including several cases of re-descrip- tions and re-allocations has been intensively debated (Johnson et al. 2001; Johnson and Wilmer 2015; Tavera et al. 2018; Damadi et al. 2020). In reality, further research on phylogenetic relationships and taxonomic treatment which, in the past, has been less identified in these genera of Haemulidae is required to figure out the actual num- ber of species because of nominal and complex species as well as the intergeneric relationships between these groups of fishes (Johnson and Wilmer 2015; Tavera et al. 2018). Moreover, many haemulid species manifest- ed morphological diversity in colouration and markings with developmental stages, for example, Plectorhinchus species, or population variation associated with changes in environmental conditions, such as Diagramma pictum (McKay 1983; Liang et al. 2016). Thus, adopting a mul- tiple analytical approach to DNA sequence data can be promising (Padial et al. 2010; Carstens et al. 2013) be- cause it evaluates the morpho-molecular diversity to un- derstand their phylogenetic relationships and lowers the possibility of homoplasy (Nei and Kumar 2000; Tavera et al. 2011; Varon-Gonzalez et al. 2020). The genera Diagramma and Plectorhinchus comprise 35 valid spe- cies worldwide (Fricke et al. 2022). Recent molecular studies confirmed that the subfamily Plectorhinchi- nae 1s monophyletic, but the phylogenetic relationships amongst some genera are challenged by those retrieved by morphological studies (Sanciangco et al. 2011; Tavera et al. 2018). Johnson et al. (2001) examined the genus Diagramma and five geographically constrained subspe- cies recognised within D. pictum (Thunberg, 1792), based ona morphological approach: D. p. pictum from Indo-Ma- lay Archipelago to Central Indo-Pacific excluding Austra- lia (type locality: Japan); D. p. labiosum from northern Australia to South New Guinea (type locality: Australia, Queensland); D. p. cineraseens from the Persian Gulf to the Bay of Bengal (type locality: Sri Lanka, Trincomalee); D. p. punctatum from the Red Sea (type locality: north- ern Red Sea) and D. p. centurio occurring along the African coast (type locality: Seychelles). The taxonom- ic position of Digramma members has been challenged (Allen 1997; Johnson et al. 2001; Heemstra and Heemstra 2004; Bogorodsky et al. 2014; Johnson and Wilmer 2015; Bray 2017; Golani and Fricke 2018). Recently, many subspecies have been promoted to the status of species rank (Fricke et al. 2022). After examining several spe- cies, Tavera et al. (2018) proposed to classify them under Plectorhinchus, but it is still necessary to investigate the remaining nominal species (D. labiosum Macleay, 1883, D. punctatum Cuvier, 1830 and D. melanacrum Johnson & Randall, 2001). Of 42 species, 11 species are morpho- logically known from the northwest Indian Ocean, includ- ing Diagramma pictum, D. punctatum, Plectorhinchus albovittatus (Ruppell 1838), P flavomaculatus (Cuvier, 1830), P playfairi (Pellegrin, 1914), P gaterinus (Forsskal, 1775), P. gibbosus (Lacepéde, 1802), P. pictus (Tortonese, 1936), P. sordidus (Klunzinger, 1870), P. schotaf (Forsskal, 1775) and P. makranensis Damadi, zse.pensoft.net Damadi, E et al.: Species delimitation, molecular phylogeny of the sweetlips Yazdani Moghaddam, Ghassemzadeh & Ghanbarifar- di, 2020. A comprehensive analysis of all species in this region has not been done. Some of these species have limited distribution, such as D. punctatum (Red Sea) and Plectorhinchus makranensis (the Persian Gulf to the Gulf of Oman) (Zajonz et al. 2019; Damadi et al. 2020). In addition, few genetic studies have explored haemulid species, including DNA barcoding analysis in a few taxa from the northwest Indian Ocean (Asgharian et al. 2011; Rabaoui et al. 2019). The main goals of this study are as follows: (1) to test the validity of the northwest Indian Ocean taxa proposed by morphological classification, (2) to utilise the wide geographic coverage of sweetlips spe- cles using species-delimitation algorithms to shed light on their molecular operational taxonomic units (MOTUs), (3) to reconstruct robust phylogenetic trees using two mtDNA (COI and Cyt 6 and one ncDNA (RAGI1) markers to infer the phylogenetic relationships and the systematic position of Diagramma pictum and D. punctatum and (4) to estimate divergence times between genera and species and reconstruct ancestral ranges to test the relative impor- tance of historical geology, biology and ecological factors for the diversification within major clades of the subfam- ily Plectorhinchinae. Materials and methods Sample collection and specimen examination A total of 254 individuals belonging to 29 species, in- cluding both our new samples and the sequences taken from GenBank and the Barcode of Life Data systems (BOLD systems) were analysed (Suppl. material 1). A to- tal of 44 new sequences of three molecular markers were used. The specimens were photographed and then a small piece of right pectoral fin of each individual was excised and preserved in absolute alcohol. The whole samples were stored in formaldehyde for long-term storage. All collected fish specimens were identified as described by Randall (1995). Voucher specimens are deposited in the Zoological Museum, Ferdowsi University of Mashhad (ZMFUM), Iran (Suppl. material 1). DNA extraction, amplification and sequencing Genomic DNA was extracted from the muscle tissue following the BioGene kit protocol. The reactions volume of polymerase chain reaction (PCR) was 25 ul, including 2 ul template DNA, 9.5 yl ddH,O, 12.5 pl Master Mix and 0.5 ul of each forward and reversed primer with 10 uM concentration. Two mitochondrial and one nuclear gene were amplified as follows: 617 bp of the CO1 was ampli- fied using universal primers COILBC_F and COIHBC_R (Ward et al. 2005), 1100 bp of the Cyt 6 was amplified using UniF and UniR primers (Orrell et al. 2002) and 1440 bp of the nuclear gene RAG1 was amplified using nested PCRs Zoosyst. Evol. 99 (1) 2023, 135-147 product (1** PCR: 2510F and RAGIR1; 2"? PCR: RAGIF1 and RAGIR2 primers) (Lopez et al. 2004). PCR conditions for the COI, Cyt 6, and Ragl genes were as reported by Sanciangco et al. (2011). The PCR products were evaluat- ed with 1.5% agarose gel for band characterisation, puri- fied and then sent for sequencing at Microsynth Company, Switzerland. Ocyurus chrysurus (Bloch, 1791) and Rhom- boplites aurorubens (Cuvier, 1829) from the family Lut- janidae Gill, 1861 were used as outgroups, since Lutjanid species has a close phylogenetic relationship with Haemul- idae (Betancur-Rghton et al. 2013; Near et al. 2013). Phylogenetic analysis and population structure The quality of new sequences was probed using the CLC Genomics Workbench v.3.6.5 software (QIAGEN Bioinformatics). The sequences were aligned in MAFFT v.7.463 and edited with BIOEDIT v.7.2 software (Hall 1999). Genetic interspecific distances of the mitochondrial COI gene were calculated by MEGA X (Kumar et al. 2018) using the Kimura two-parameter (K2P) model. The best-fit nucleotide substitution model was identified by the Akaike Information Criterion (AIC) in the jModelTest v.2 (Posada 2008). AIC was TIM2+I+G for COI, GTR+I+G for Cyt b, GTR+I+G for the two mtDNA regions and TIM2+I+G for the mitochondrial and nuclear markers. Maximum Likelihood (ML) analysis was performed using RAXxMLy.7.2.6 (Stamatakis 2014) with 1000 replicate bootstrap. Maximum Likelihood (ML) analysis was performed using RAxMLv.7.2.6 (Stamatakis 2014) with 1000 replicate bootstrap. Bayesian Inference (BI) was run in MrBayes v.3.2.7 (Ronquist et al. 2012) with two independent Markov Chain Monte Carlo for 50,000,000 generations. The first 25% of trees were discarded as part of the burn-in phase. The remaining trees were combined to generate a 50% majority rule consensus tree. The posterior probability and bootstrap values were calculated from the BI and ML for each clade and values above 0.95 and 75 were deemed as significant supports, respectively. A haplotype network was constructed using the median- joining method in the PopART v.1.7 package (Leigh and Bryant 2015). The parsimony informative and variable sites, as Well as haplotype diversity were calculated using DnaSP v.5.10 to test patterns of population structure in D. pictum and D. labiosum (Librado and Rozas 2009). Initially, genetic divergence between the putative species of Diagramma was estimated and the analysis of molecular variance (AMOVA) as ®,, values was performed in GenAIEx v.6.5 (Peakall and Smouse 2006) based on pairwise comparisons. Demographic changes in the effective population size were investigated for both Species using two neutrality tests (Fu’s Fs and Tajima’s D) and the Ramos-Onsins and Rozas test R2 in DnaSP v.5.10 (Librado and Rozas 2009). The time elapsed since most recent population expansion was calculated herein for Diagramma pictum and D. labiosum (Rogers and Harpending 1992) using the t = 2ut value, where t 137 is the time elapsed since the start of expansion in years, u is the mutation rate per generation for the sequence (the number of base pairs per divergence rate (1% or 2% Mya). generation time (in years) and t is the time elapsed since the beginning of expansion in years. As reported by Grandcourt et al. (2011), the generation time is 2.9 years for Diagramma pictum. The mutation rate reported for the mtDNA (COI) in previous reef fish studies ranged from 1% to 2% per million years (Bowen et al. 2001). Divergence time estimation A calibrated time-tree of Plectorhinchinae was estimat- ed from the combined data set (COI, Cyt 6 and RAGI: 3157 total bp) in BEAST v.2.6.2 (Bouckaert et al. 2019). Three independent MCMC chains were run for 20 million generations each with sampling every 1,000 generations. Runs were implemented using an uncorrelated lognormal relaxed clock model to calculate rate heterogeneity and the Yule prior method for the tree prior. Convergence of runs and the effective sample size (> 200) were checked in Tracer v.1.6 (Rambaut 2016). The three runs were combined by LogCombiner v.2.6.2 (Bouckaert et al. 2019). The first 25% of trees were discarded in the Tree- Annotator v.2.6.2 (Bouckaert et al. 2019). The consensus tree with divergence time estimates was visualised using FigTree v.1.4.4 (Rambaut, 2016). Due to a lack of fossil record for Haemulidae, two calibration points were used following Near et al. (2013) and Tavera et al. (2018): (1) the Haemulidae-Lutjanidae split in early Eocene (mean 52.5, 95% HPD interval 57.2-46.8) and (2) the Para- pristipoma-Plectorhinchus split in the late Eocene (mean 34.5 Ma, 95% HPD interval 42.6—27 Ma). Ancestral area reconstruction The ancestral area in Plectorhinchinae was inferred using the BioGeoBEARS analysis in RASP v.4 (Yu et al. 2020). Geographic distribution of each species was derived from literature (Fricke et al. 2022) and sampling of our rang- es (Suppl. material 1). Four biogeographic regions were defined, based on present-day distribution and genetic diversification reported in previous studies (Ma et al. 2016; Stiller et al. 2022): Western Atlantic (WA); Eastern Atlantic (EA); Western Indian Ocean (WIO); Central In- do-Pacific (CIP). The input trees of BEAST analysis were used to infer the ancestral area probability. Six different biogeographic models were evaluated to select the best- fit using the AIC cumulative weight (AICc_wt). Molecular species delimitation Molecular species delimitation was used from four dif- ferent methods, including; (A): the Automatic Barcode Gap Discovery (ABGD) (Puillandre et al. 2012) uses the zse.pensoft.net 138 pairwise genetic distance to detect a barcode gap while as- suring that intra and interspecific genetic distances do not overlap and are executed in the website (http://wwwabi. snv.jussieu.fr/public/abgd) with prior intraspecific di- vergence (0.001 to 0.1), steps = 10 and a relative gap width of 1 using the Kimura 2-parameter model (Kimura (K80)); (B): Assemble Species by Automatic Partitioning (ASAP) (Puillandre et al. 2021), based on the distance threshold and a hierarchical clustering algorithm to cre- ate a list of best partitions. The partition score combines two metrics: the barcode gap width and the probability of panmixia (p-value), in which a lower score indicates a better partition. The method that was done using the website https://bioinfo.mnhn.fr/abi/public/asap with the Kimura (K80) model; (C): Bayesian Poisson Tree Pro- cess (bPTP) (Zhang et al. 2013), a method that estimates the number of mutations between two branching events using the branch length information of the input phyloge- ny. The bPTP approach was adopted on its website (http:// species. hits.org/ptp/) using a non-ultrametric tree of ML inference as the input, 500,000 MCMC generations and 10% burn-in; (D): Generalised Mixed Yule Coalescent (GMYC) (Pons et al. 2006) method was run on its web- site (http://species.h-its.org/gmyc), using the ultrametric tree produced by BEAST v.2.6.2 (Bouckaert et al. 2019). Results MOTU delimitation and genetic diversity We assembled a total of 217 COI sequences (34 newly developed plus 183 archived; 617 bp) from 29 nominal Plectorhinchinae species. D. punctatum and Plectorhinchus pictus were sequenced for the first time. Novel COI barcodes were provided for Plectorhinchus flavomaculatus, P. gaterinus and P. gibbosus for the first time from the Northern Indian Ocean (NIO). Of 29 nominal species studied, the coalescent-based delimitations (bPTP, GMYC) yielded 27 MOTUs, whereas the distance-based methods (ABGD and ASAP) yielded 26 MOTUs (Fig. 1). ABGD and ASAP identified Plectorhinchus lessonii, and P. diagrammus and P. albovittatus and P. caeruleonothus as a MOTU. bPTP and GMYC methods considered Plectorhinchus lessonii and P. diagrammus as one MOTU. Additionally, haplotype networks, based on the COI gene, identified two main distinct haplogroups (Fig. 2A). The haplogroup I was composed of Diagramma pictum from the NIO to WP, which was separated by 15 mutation steps from haplogroups II, represented by D. punctatum from the Red Sea. In the first haplogroups, samples of D. pictum were mixed with haplotypes of D. Jabiosum from Australia. As suggested by the genetic distance method that uses the K2P model, the highest interspecific genetic distance was found between Plectorhinchus macrolepis and P. schotaf (21.3%). In comparison, the lowest distance was observed between D. pictum and D. labiosum (0.53%) and P. lessonii and P. diagrammus (1.2%) (Table 1). The zse.pensoft.net Damadi, E et al.: Species delimitation, molecular phylogeny of the sweetlips Table 1. Genetic K2P distances (%) of the Diagramma and Plectorhinchus species. Species Maximum intra- Nearest- Distance group distance Neighbour (NN) to NN Diagramma pictum 0.17 D. labiosum 0.53 D. punctatum 0.00 P. centurio 3.45 Plectorhinchus 0.00 P. caeruleonothus 25a albovittatus P. caeruleonothus 0.19 P. albovittatus Ask P. centurio 0.35 D. punctatum 3.45 P. chaetodonoides 0.43 P. centurio 8.75 P. chrysotaenia 0.00 P. playfairi 16.30 P. chubbi 0.08 P. sordidus B52 P. cinctus 0.64 P. gibbosus 13.44 P. diagrammus 0.00 P. lessonii 1.20 P. flavomaculatus 0.31 P. makranensis 12.62 P. gaterinus 0.28 P. caeruleonothus 12.18 P. gibbosus 0.69 P. cinctus 13.44 P. lessonii 0.08 P. diagrammus 1.20 P. lineatus 0.06 P. polytaenia 7.17 P. macrolepis 0.00 P. plagiodesmus 13.84 P. macrospilus 0.00 P. lessonii 4.10 P. makranensis 0.01 P. schotaf owas: P. orientalis - P. vittatus 7.97 P. pictus 0.00 P. cinctus 13:55 P. picus 0.08 D. punctatum 8.38 P. plagiodesmus 0.00 P. pictus 13.24 P. playfairi 0.16 P. chubbi 4.61 P. polytaenia 0.00 P. lineatus Pairs P. schotaf 0.13 P. makranensis 523 P. sordidus 0.10 P. chubbi 3.52 P. unicolor 0.23 P. chubbi 5.80 P. vittatus - P. orientalis 7.97 highest intraspecific genetic variation was observed in P. gibbosus (0.69%), while values close to 0 to 0.09% were detected in most of the Diagramma and Plectorhinchus species (Table 1). Based on all delimitation analyses, D. labiosum specimens from Queensland, Australia (type locality) are genetically analogous to D. pictum and there is no evidence of their genetic differentiation. Cyt b sequence of a single D. Jabiosum specimen (DQ784750), deposited at the GenBank of Australia with D. pictum specimens in the phylogenetic tree, was identified as a monophyletic lineage (genetic divergence: 0.51%) (data are not shown). Seventeen haplotypes were detected based on COI sequences between D. labiosum and D. pictum (Table 2). Seventeen haplotypes were detected, based on COI sequences between D. labiosum and D. pictum (Table 2). The highest haplotype and nucleotide diversity indices were found in the samples of D. /abiosum (Australia) (Hd = 0.600, 2 = 0.0021), while specimens of D. pictum (Hd =0.560: NIO, 7=0.0018: WP) demonstrated the lowest values (Hd = 0.579, 7 = 0.001) (Table 2), where the diversity indicators were not influenced by sample size reduction. OST value revealed a non-significant level between Australia (D. labiosum) and IWP, except Australia (D. pictum) (®,. = —1.783) (Table 2). Test of neutrality found negative and non-significant Tajima’s D values, while Fu’s Fs (-8.801) and R2 (0.038) results were significant (Table 2). These significant values (Fu’s Zoosyst. Evol. 99 (1) 2023, 135-147 0.2 SRR eee cD 139 P. flavomaculatus P. makranensis P. schotaf P. playfairi P. sordidus P. chubbi P. unicolor P. chrysotaenia P. gaterinus P. caeruleonothus P. albovittatus D. pictum,D. labiosum P. centurio D. punctatum > chaetodonoides P. lessonii, P. diagrammus P. macrospilus P. polytaenia P. lineatus P. orientalis P. vittatus P. gibbosus P. plagiodesmus P. macrolepis P. pictus P. cinctus SRR RRR eee As BERR eee ie BERR RR coc Figure 1. Bayesian Inference tree, based on the COI showing the results of species delimitation algorithms. The vertical bars in- dicate species delimitation analyses. The black circles indicate nodes with supports (ML bootstrap BP > 75% and BI probability PP => 0.95), orange circles (BP < 75% and BI PP = 0.95) and blue circles (PP < 0.95 and ML BP < 75%). Fs, R2), in combination with the estimates of t for both Species, indicate a shallow population history with recent expansion (Table 2). The unimodal mismatch distribution for the pooled sample of D. pictum supported a recent historical expansion (Fig. 3). Phylogenetic relationships We analysed 254 COI DNA sequences (617 bp) which show 325 variable sites; the Cyt b gene consisting of 1117 bp showed 295 variable positions; and Rag1 consisting of 1440 bp showed 184 variable sites. Neither gaps nor stop codons were observed in the final alignment. ML and BI analyses with the combined dataset (mtDNA and nuclear) revealed that the phylogenetic relationships of sweetlips species had the highest resolution (Fig. 4). The ML and BI phylogenetic trees, based on two mtDNA (Cyt b + COJ) and one nuclear (Ragl) gene, had a congruent topology, with a slight difference in their support values. The phylogenetic relationships observed in the analysis of the concatenated data (Cyt b + COI + Rag1) revealed that Parapristipoma, as a basal position, is the sister clade of the Plectorhinchus species with high bootstrap and posterior probabilities (Fig. 4). Within the Plectorhinchus clade, two well-supported subclades were formed: (1) The first clusters the most WIO species (subclade I): Plecto- rhinchus schotaf, P. makranensis, P. playfairi, P. sordi- dus, P. chubbi, P. unicolor, P. plagiodesmus, P. gibbosus, P. macrolepis, P. chrysotaenia, P. cinctus and P. pictus; (2) the second clusters the most CIP species: P. gaterinus, P. chaetodonoides, P. picus, P. albovittatus, P. lessonii, P. lineata, P. macrospilus, P. diagrammus, P. polytaenia, P. vittatus, P. orientalis, P. caeruleonothus along with D. pictum and D. punctatum indicating that the Diagram- ma species is nested within Plectorhinchus and should be recognised as Plectorhinchus pictum and Plectorhinchus punctatum (subclade IT) (PP = 1; BS = 100%). zse.pensoft.net 140 Haplogroup II Haplogroup I Damadi, E et al.: Species delimitation, molecular phylogeny of the sweetlips Figure 2. Median-joining network obtained of 617 bp of mitochondrial COI for Diagramma species (A). Map depicting the geo- graphical distribution of 17 haplotypes (B). Circle sizes are relative to haplotype frequency. Numbers between haplotypes represent mutational steps between haplotypes. Colour circles indicate species of D. pictum (red: Persian Gulf to Bangladesh, green: China and Indonesia) and D. /abiosum (orange: Australia), D. punctatum (black: Red Sea). Observed \ tomo Expected i 03 \ e \ = \ g \ = 02 \ o 4 - 1 ia \ \ 0.1 \ 3 * \ c oe o ____ i a ied + aot + 0 5 10 15 20 Pairwise differences Figure 3. Mismatch distribution inferred from partial mtDNA COI for the pooled dataset of D. pictum. Divergence times and ancestral area reconstructions The Haemulidae diverged from Lutjanidae ~ 49 Ma (ear- ly to middle Eocene, 95% HPD: 43.6—55.2 Ma) and then Plectorhinchinae divided into two main clades (A-—B) and two subclades (I-II) (Fig. 5). The first split created Clade A containing Parapristipoma at ~ 35 Ma (late Eo- cene, 95% HPD: 32.4—37.1 Ma). Clade B diverged at ~ 21 Ma (early Miocene, 95% HPD: 18.4—23.5 Ma) into two subclades. Within each subclade, there were two pe- riods of maximum diversification from the Miocene to zse.pensoft.net Table 2. Molecular diversity indices for D. pictum and D. labio- sum based on mitochondrial DNA (COI) sequence data. Numbers in bold denotes significance (P< 0.05) and for Fu’s Fs (P< 0.02). Statistics D. pictum D. labiosum Japan to Iran to Australia All Indonesia Bangladesh (WP) samples (WP) (NIO) Number of individuals (N) N = 44 N = 26 N=/7 N = 76 Expansion time (years) _ - - 17,647- 35,294 Number of haplotypes (Hn) 8 6 3 17 Haplotype diversity 0.579 + 0.560 + 0.600+ 0.624+ (Hd + SD) 0.075 0.073 0.215 0.051 Nucleotide diversity 0.0018+ 0.0021+ 0.0021+ 0.0024 + (7 + SD) 0.0011 0.0016 0.0017 0.0018 Tajima’s D -1.218 -1.636 -1.233 -1.783 Fu’s FS -3.739 -1.067 0.189 -8.801 R2 0.058 0.060 0.062 0.038 O., Australia 0.084 0.072 = lran to 0.049 - - Bangladesh Pliocene epochs. The best-fit biogeographic models re- covered for the ancestral range reconstruction was the BAYAREALIKE (AICc_wt: 0.71). As suggested by the ancestral area reconstruction using the combined dataset (Cyt b + COI + Ragl1), 17 dispersal and eight vicariance events occurred during the evolution of the studied Plec- torhinchinae. The deepest node of early Plectorhinchinae constructed probably has its origin in the [TWP with the dispersal and vicariance events occurring from the late Eocene to early Oligocene epoch (Fig. 6). However, more comprehensive taxonomic sampling of the Parapristipo- ma species can help find a definitive point for the origin Zoosyst. Evol. 99 (1) 2023, 135-147 P. makranensis - P schotaf P. flavomaculatus P. sordidus se P chubbi P. playfairi P. unicolor P. chrysotaenia P pictus P. plagiodesmus P. macrolepis P. gibbosus P. cinctus D. pictum P. centurio D. punctatum FP. chaetodonoides P. picus P. lessonii e P diagrammus P. macrospilus P. vittatus P. orientalis P. lineatus P. polytaenia P. gaterinus P. albovittatus P. caeruleonothus P. octolineatum Subclade I Subclade II 141 Ocyurus chrysurus P. trilineatum Rhomboplites aurorubens 0.04 Figure 4. Phylogeny recovered by the Bayesian Inference (BI) and Maximum Likelihood (ML) analyses for the Cyt b, COI and RAGI dataset. The black circles indicate nodes with supports (ML bootstrap BP = 75% and BI probability PP = 0.95), orange circles (BP < 75% and BI PP = 0.95) and blue circles (PP < 0.95 and ML BP < 75%). of the subfamily Plectorhinchinae. The ancestors of the earliest Plectorhinchus lineages were supposed to be of WIO origin with vicariance and dispersal events taking place in the Miocene. Within subclade I, there seems to be a burst of species diversification in the WIO at 20 Mya. Within subclade II, the vicariance event affected specia- tion in the CIP as the ancestral area at 15 Mya. Discussion This study represents the first attempt to delimit the species boundaries and evolutionary and biogeographic history of marine fish species in Plectorhinchinae. The present study corroborates the findings of the previous molecular studies that show paraphyly of the Plecto- rhinchus without the inclusion of Diagramma (San- ciangco et al. 2011; Tavera et al. 2018) and contrast with morphological data (Konchina 1976; Carpenter and Niem 2001; Nelson et al. 2016) that supported this genus monophyly. Molecular phylogeny of Plectorhinchus According to the molecular data, Plectorhinchus is divid- ed into two subclades (Fig. 4), which display high concor- dance with the division proposed by Tavera et al. (2018). It can reinforce the phylogenetic value of the geographic dis- tribution and body-colour pattern in distinguishing P/ecto- rhinchus lineages. The first group (subclade I) mostly con- sists of species with uniformly dark colours (Plectorhinchus schotaf, P. makranensis, P. playfairi, P. sordidus, P. chub- bi, P. unicolor, P. plagiodesmus, P. gibbosus, P. macrolepis and P. chrysotaenia) or black pots on the upper sides of the body (P. cinctus and P. pictus), along with more profound body depth (Randall 1995, 1997; McKay 2001), which are those typically distributed in the WIO (Fig. 4) (Fricke et al. 2022). The second group (subclade II) was comprised of species with spots on the whole body (P. gaterinus, P. cha- etodonoides and P. picus) or horizontal stripes alongside (P. albovittatus, P. lessonii, P. lineata, P. macrospilus, P. diagrammus, P. polytaenia, P. vittatus and P. orienta- lis), except for P. caeruleonothus and Diagramma species zse.pensoft.net 142 Damadi, E et al.: Species delimitation, molecular phylogeny of the sweetlips D. pictum P centurio D. punctatum P. chaetodonoides PB picus P. vittatus P. orientalis P. lineatus P. polytaenia ' EL lessonii P. diagrammus P. macrospilus | 2 gaterinus | P. albovittatus iP 2 caeruleonothus P. makranensis a 4 P. schotaf P. flavomaculatus P. sordidus 1 & chubbi 1 P playfairi P. unicolor 1) PB chrysotaenia P. pictus | 2: plagiodesmus P. gibbosus P. cinctus | B. macrolepis PB trilineatum P. octolineatum Ocyurus chrysurus | Rhomboplites aurorubens Miocene 50 40 30 20 10 0 Figure 5. Time-calibrated phylogeny, based on combined mitochondrial (COI, Cyt 5) and nuclear (RAG1) genes, inferred from the BEAST analysis of the Plectorhinchinae. Numbers in the horizontal purple bars represent the 95% Highest Posterior Density inter- vals for each estimated event. The black circles (pp = 0.95) and orange circles (pp < 0.95). A: Parapristipoma, B: Plectorhinchus. (Fig. 4) (Randall 1995, 1997; Johnson et al. 2001; McKay 2001). The species includes representatives with shorter bodies from the CIP (Fricke et al. 2022). The colour varia- tion observed in subclade II compared to subclade I can be attributed to the substrate type (Randall et al. 1997; Mck- ay 2001; Johnson et al. 2001). The colour variation of the members of subclade II inhabiting the coral reef may be linked to social communication, mimicry and camouflage defence mechanisms to avoid predation adjusted to their habitat (Randall 1998; McMillan et al. 1999). Systematics and species boundaries of Plectorhinchus In our analyses, specimens from 29 nominal species were delimited between 26 and 27 consensus MOTUs. In gen- eral, low interspecific genetic divergences were observed in some of the MOTUs within Plectorhinchus, which focuses on the barcoding analysis in closely-related spe- cies. This situation was formerly reported in haemulid within Anisotremus (Bernardi et al. 2008) and Haemu- Jon (Rocha et al. 2008). In the meanwhile, the fact that P. lessonii and P. diagrammus, along with P. albovittatus and P. caeruleonothus, grouped together in all the delim- itation analyses, exhibits their very recent divergence (< 1 Ma) compared to other Plectorhinchus members. The morphological variability, including minor differences in zse.pensoft.net colouration between D. pictum and D. labiosum deter- mines taxonomic classification into subspecies or species status (Allen 1997; Johnson et al. 2001; Bogorodsky et al. 2014; Bray 2017; Fricke et al. 2022). Our molecular analyses did not yield any genetic evidence to confirm D. labiosum as a distinct species (Figs 1, 2, Table 1). A rela- tively low level of genetic divergence between D. pictum and D. labiosum found in both markers (COI and Cyt 5) may indicate a recent speciation event (Table 1). These distances are considerably shorter than those reported in haemulid species (Johnson and Wilmer 2015; Damadi et al. 2020) and other related species of marine fishes, which is at least 2% between closely-related species (Ward et al. 2005; Hsu et al. 2007). Demographic tests (Fu’s, R2 and mismatch distribution) indicate that D. Jabiosum and D. pictum share a common ancestor and expansion events over the last few thousand years coincided with the Ho- locene glaciations that restricted larval dispersal and ocean circulation deflection (Yokoyama et al. 2001; Wil- son 2013; Rosser et al. 2020). These features might have reduced coral reef habitats and colonisation from north- west Australia, especially in the reef fish (Ovenden et al. 2002; Salini et al. 2006; Sulaiman and Ovenden 2010). The incongruence between colour variation and the lev- el of genetic divergence is not rare in fish and could be attributed to some other reasons: (1) phenotypic plastici- ty rather than reproductive isolation, (2) incomplete lin- eage sorting and (3) introgression through hybridisation Zoosyst. Evol. 99 (1) 2023, 135-147 e & @ e @ | Western Atlantic (WA) [| Eastern Atlantic (EA) © Sl Western Indian Ocean (WIO) ° WN central Indo-Pacific (CIP) indo West Pacific (WP) e e e @ 143 Plectorhinchus centurio Diagramma pictum Diagramma punctatum Plectorhinchus chaetodonoides Plectorhinchus picus Plectorhinchus vittatus Plectorhinchus orientalis Plectorhinchus lineatus Subclade IT Plectorhinchus polytaenia Plectorhinchus lessonii Plectorhinchus diagrammus Plectorhinchus macrospilus Plectorhinchus gaterinus Plectorhinchus albovittatus Plectorhinchus caeruleonothus Plectorhinchus makranensis Plectorhinchus schotaf Plectorhinchus flavomaculatus e Plectorhinchus sordidus Plectorhinchus chubbi Plectorhinchus playfairi Plectorhinchus ree S ub cl ad e I Plectorhinchus chrysotaenia Plectorhinchus pictus Plectorhinchus plagiodesmus Plectorhinchus gibbosus Plectorhinchus cinctus Plectorhinchus macrolepis q Parapristipoma trilineatum Parapristipoma octolineatum Ocyurus chrysurus Rhomboplites aurorubens Figure 6. Ancestral area reconstruction obtained using BioGeoBEARS for Plectorhinchinae. Biogeographic areas are shown in the circles and the colours in the squares indicate the current species distribution. (Bowen et al. 2006; Schultz et al. 2007; DiBattista et al. 2012; DiBattista et al. 2015; Bernal et al. 2017). There are several lines of evidence that favour the incomplete lin- eage sorting over other scenarios; however, there is still a paucity of evidence and further analyses using genomic data are warranted. Firstly, D. Jabiosum and D. pictum showed extensive mtDNA sharing and coalescence of haplotypes, which could be interpreted as evidence of a very recent divergence (Figs 1, 2). Secondly, their distri- bution is allopatric (D. /abiosum: Australia and D. pic- tum: IWP, except Australia) (Fig. 2B) (Fricke et al. 2022). Hybridisation events and lineage sharing usually occur in sympatric and parapatric areas rather than in allopatric areas due to the greater opportunities for hybridisation in the secondary contact zones (Hobbs et al. 2009; DiBattis- ta et al. 2016). In contrast, incomplete lineage may take place in both sympatric and allopatric areas (DiBattista et al. 2012; DiBattista et al. 2015). Thirdly, regional colour morphs usually represent minor molecular divergences due to recent genetic divergence, which may not be re- lated to ecological and behavioural adaptations (Taylor and Hellberg 2003; Drew et al. 2008; Drew et al. 2010). Fourthly, the Diagramma species are distinguished only based on colour patterns (Johnson et al. 2001; Heems- tra and Heemstra 2004; Bogorodsky et al. 2014) which evolve more quickly than the other morphological traits and genetic characters (Bowen et al. 2006; Schultz et al. 2007). Colouration could be an important criterion to distinguish closely-related species in haemulid fishes (Johnson and Wilmer 2015; Damadi et al. 2021). Fifthly, the most recent divergence occurred between D. pictum and D. labiosum around 28—56 million years ago (Table 1), which is more recent than the complete lineage sorting time. A divergence time of 2.9 Mya seems sufficient for these lineages for complete sorting (1 generation time) (Grandcourt et al. 2011). Biogeography and temporal diversification Our biogeographic findings exhibit a complex history highlighting the role of both biological and geographical factors in the diversification and evolution within the Hae- mulids. The first diversification, 1.e. the split of Haemulids from Lutjanids as a sister lineage in the Eocene, could be related to the ice sheet formed in the Antarctic and reduced atmospheric CO, level (Zachos et al. 2001; Coxall et al. 2005; Lear et al. 2008). These climatic and oceanographic changes resulted in a sea-level drop, upwelling and extinc- tion of warmer climate fauna (Zachos et al. 2001; Liver- more et al. 2007; Ma et al. 2016). This extinction formed vacant niches and the emerging Indo-Australian Archipel- ago probably gave rise to new reef habitats and high spe- cies-richness in the cooler oceans (Cowman and Bellwood 2011; Ma et al. 2016). This divergence time corresponds to the one shown by Near et al. (2012) for the same his- zse.pensoft.net 144 torical event in Haemulids. The historical biogeography suggests that the common ancestor of Plectorhinchinae might have its origin in the [WP and subsequently spread and diversified in the western Indian Ocean and the Cen- tral Indo-Pacific. This scenario could be firmly demon- strated by complete biogeographic sampling of some taxa and extra genetic/genomic analyses. For Plectorhinchinae, divergence was first observed in East Atlantic (EA) spe- cies (Parapristipoma) with the fewest species from the Plectorhinchus \ineage, approximately 33.8 Mya, which coincided with the onset of the terminal Tethyan event (TTE) that disconnected West Tethyan and Arabian ma- rine biodiversity hotspots (Adams et al.1983; Harzhauser et al. 2007). The diminishing connectivity between two re- gions might have set the scene for vicariant splits, which appear to be linked to the early diversification of this clade. This divergence time is also congruent with the EA and IWP divergence in other species of reef fish, particularly the Groupers and Boxfishes (Santini et al. 2013; Ma et al. 2016). Within the Plectorhinchus TWP Clade, two factors, the Indo-Australian Archipelago (IAA) uplift and major global cooling, were associated with the diversification of sweetlips subclades. These factors were inferred from other reef fish in the Miocene period (Zachos et al. 2001; Bellwood and Wainwright 2002; Shevenell et al. 2004; Cowman and Bellwood 2011; Ma et al. 2016). An esti- mate of divergence times of each Plectorhinchus subclade suggests further diversification occurred during the Mio- cene that slow indicated by the long branches, followed by slower diversification in Pliocene to Pleistocene epochs (Fig. 5). The IAA uplift during the early to mid-Miocene in subclade II (CIP Plectorhinchus) created new islands, coinciding with the lowering of sea level, leading to novel shallow reef habitat discontinuity. During Plio-Pleistocene, Diagramma species diverged from each other around 4.1 to 2.3 Mya (Fig. 5) and D. punctatum, an endemic spe- cies, formed in the Red Sea, coinciding probably with high temperature and salinity, sea-level changes during glacial cycles and the narrow Strait of Bab al Mandab. These fac- tors led to a decrease in unsuitable habitat and the water flow through this strait (Rihm and Henke 1998; Riegl and Purkis 2012; Stevens et al. 2014; Torquato et al. 2019) that has been documented in marine fish (Froukh and Kochzius 2007; DiBattista et al. 2013; Bogorodsky et al. 2017). The major global cooling could lay the ground for emergence of new environmental niches likely driven by adaptive ra- diations in subclade I (WIO) during the Miocene (Zachos et al. 2001; Shevenell et al. 2004). Conclusions The findings of the present study on _ sweetlips (Plectorhynchinae) not only shed light on the interrelation- ships and evolutionary history, but also opened new avenues for research. The results of this study provide a framework to shed light on the probable origin and factors involved in the diversification of the [WP Plectorhynchinae. The contemporary species have a common ancestor that comes zse.pensoft.net Damadi, E et al.: Species delimitation, molecular phylogeny of the sweetlips from the present day [WP and the current geographical dis- tribution appears to be related to vicariance and dispersal events. It indicates that Diagramma species were divided 4.1 to 2.3 Mya ago into ancestral Diagramma punctatum in the Indian Ocean, directly followed by the WP and re- cently isolated species. These findings raise the possibility that recent genetic divergence have given rise to colour morphotypes as emerging species on coral reefs. Acknowledgements We greatly thank Dr. Joseph D. DiBattista and Dr. Brian W. Bowen for their valuable comments on population ge- netics. The authors are especially grateful to Mohamed Awad for providing tissue specimens from Red Sea (Egypt). We would like to thank local fishermen for their help in collecting specimens. 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Bioinformatics 29(22): 2869-2876. https://doi.org/10.1093/bioin- formatics/btt499 Supplementary material | Sampling information and GenBank accession numbers for the specimens included in the phylogenetic analyses Authors: Ehsan Damadi, Faezeh Yazdani Moghaddam, Mehdi Ghanbarifardi Data type: table (excel file) Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zse.99.96386.suppl 1 zse.pensoft.net