Portal de Periódicos da CAPES

Systematics, genetics, and biogeography of intertidal mites (Acari, Oribatida) from the Andaman Sea and Strait of Malacca

2018; Wiley; Volume: 57; Issue: 1 Linguagem: Inglês

10.1111/jzs.12244

1439-0469

Tobias Pfingstl, Andrea Lienhard, Satoshi Shimano, Zulfigar Yasin, Aileen Tan Shau Hwai, Sopark Jantarit, Booppa Petcharad,

Insect-Plant Interactions and Control

Journal of Zoological Systematics and Evolutionary ResearchVolume 57, Issue 1 p. 91-112 ORIGINAL ARTICLEOpen Access Systematics, genetics, and biogeography of intertidal mites (Acari, Oribatida) from the Andaman Sea and Strait of Malacca Tobias Pfingstl, Corresponding Author Tobias Pfingstl tobias.pfingstl@uni-graz.at orcid.org/0000-0002-0778-8051 Institute of Biology, University of Graz, Graz, Austria Correspondence Tobias Pfingstl, Institute of Biology, University of Graz, Universitaetsplatz 2, 8010 Graz, Austria. Email: tobias.pfingstl@uni-graz.atSearch for more papers by this authorAndrea Lienhard, Andrea Lienhard Institute of Biology, University of Graz, Graz, AustriaSearch for more papers by this authorSatoshi Shimano, Satoshi Shimano Science Research Center, Hosei University, Fujimi, Chiyoda-ku, Tokyo, JapanSearch for more papers by this authorZulfigar Bin Yasin, Zulfigar Bin Yasin Centre For Marine and Coastal Studies, Universiti Sains Malaysia, Penang, MalaysiaSearch for more papers by this authorAileen Tan Shau-Hwai, Aileen Tan Shau-Hwai School of Biological Sciences, Universiti Sains Malaysia, Penang, MalaysiaSearch for more papers by this authorSopark Jantarit, Sopark Jantarit Excellence Center for Biodiversity of Peninsular Thailand, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, ThailandSearch for more papers by this authorBooppa Petcharad, Booppa Petcharad Department of Biotechnology, Faculty of Science and Technology, Thammasat University, Khlong Luang District, Pathum Thani, ThailandSearch for more papers by this author Tobias Pfingstl, Corresponding Author Tobias Pfingstl tobias.pfingstl@uni-graz.at orcid.org/0000-0002-0778-8051 Institute of Biology, University of Graz, Graz, Austria Correspondence Tobias Pfingstl, Institute of Biology, University of Graz, Universitaetsplatz 2, 8010 Graz, Austria. Email: tobias.pfingstl@uni-graz.atSearch for more papers by this authorAndrea Lienhard, Andrea Lienhard Institute of Biology, University of Graz, Graz, AustriaSearch for more papers by this authorSatoshi Shimano, Satoshi Shimano Science Research Center, Hosei University, Fujimi, Chiyoda-ku, Tokyo, JapanSearch for more papers by this authorZulfigar Bin Yasin, Zulfigar Bin Yasin Centre For Marine and Coastal Studies, Universiti Sains Malaysia, Penang, MalaysiaSearch for more papers by this authorAileen Tan Shau-Hwai, Aileen Tan Shau-Hwai School of Biological Sciences, Universiti Sains Malaysia, Penang, MalaysiaSearch for more papers by this authorSopark Jantarit, Sopark Jantarit Excellence Center for Biodiversity of Peninsular Thailand, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, ThailandSearch for more papers by this authorBooppa Petcharad, Booppa Petcharad Department of Biotechnology, Faculty of Science and Technology, Thammasat University, Khlong Luang District, Pathum Thani, ThailandSearch for more papers by this author First published: 04 September 2018 https://doi.org/10.1111/jzs.12244Citations: 14 Contributing authors: Andrea Lienhard (andrea.lienhard@uni-graz.at); Satoshi Shimano (sim@hosei.ac.jp); Zulfigar Bin Yasin (zulfigarusm@gmail.com); Aileen Tan Shau-Hwai (aileen@usm.my); Sopark Jantarit (fugthong_dajj@yahoo.com); Booppa Petcharad (zigzagargiope@yahoo.com) AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract This study demonstrates for the first time the presence of marine-associated mites in the Andaman Sea and Strait of Malacca and reveals a relatively high diversity of these taxa with six species from two different families: Selenoribatidae and Fortuyniidae. Indopacifica, a new genus of Selenoribatidae, is described from Thailand and Malaysia, with two new species, Indopacifica pantai n. sp. and Indopacifica parva n. sp. The genus is characterized by the unique combination of following characters: lacking lamellar ridges, incomplete dorsosejugal suture, fourteen pairs of notogastral setae, and presence of epimeral foveae. A phylogenetic reconstruction based on 18S ribosomal RNA sequences clearly confirms the distinctness of the new genus Indopacifica and places it close to the genus Rhizophobates. The lack of molecular genetic data of possible relatives impedes a clear assessment, and hence, we emphasize the need for further combined approaches using morphological and molecular genetic sequence data. All species show wide distribution areas within this geographic region suggesting that these taxa are good dispersers despite their minute size and wingless body. Molecular genetic data demonstrate recent gene flow between far distant populations of I. pantai n. sp. from the coasts of Thailand and two islands of Malaysia and hence confirm this assumption. The seasonally changing surface currents within this geographic area may favor hydrochorous dispersal and hence genetic exchange. Nevertheless, morphometric data show a slight trend to morphological divergence among the studied populations, whereas this variation is suggested to be a result of genetic drift but also of habitat differences in one population of Alismobates pseudoreticulatus. 1 INTRODUCTION The Andaman Sea is an active back-arc basin where the Indian and the Burma plate collide, and therefore, it is an area of increased seismic activities (Radhakrishna, Lasitha, & Mukhopadhyay, 2008). This area was also strongly affected by the marine earthquake of the December 26, 2004, which caused a tragic tsunami event costing thousands of lives and devastating coastal environments of the whole Indo-Pacific area. Despite this catastrophic impact which also resulted in a measurable loss of biodiversity (e.g. Adger, Hughes, Folke, Carpenter, & Rockstrom, 2005; Kumaraguru, Jayakumar, Jerald Wilson, & Ramakritina, 2005), the Andaman Sea and adjacent regions are one of the most biodiversity-rich regions and are important biodiversity hot spots (Myers, Mittermeier, Mittermeier, da Fonseca, & Kent, 2000). Moreover, the true inventory of coastal and marine biodiversity could be several times higher than what is known today as only reports of commercially important groups, for example, fishes or molluscs are very detailed but they are scarce with respect to the minor phyla (Venkataraman & Wafar, 2005). Indeed, intertidal oribatid mites are one of these minor groups and the reports from this geographic area remain poorly known, only records of three species from the nearby region of Singapore were recently published, namely Alismobates pseudoreticulatus Pfingstl, 2015, Fortuynia smiti Ermilov, Tolstikov, Mary, & Schatz, 2013 and Selenoribates asmodeus Pfingstl, 2015 (Pfingstl, 2015a,b; Pfingstl & Schuster, 2014). These tiny animals belong to the superfamily of Ameronothroidea, containing the Ameronothridae, Podacaridae, Fortuyniidae, and Selenoribatidae. The Ameronothridae and Podacaridae are exclusively occurring in cold-temperate and polar areas, and the Fortuyniidae and Selenoribatidae, on the other hand, are restricted to the subtropical and tropical zones (e.g. Pfingstl, 2017). They are air-breathing arthropods having evolved a littoral lifestyle, now leading a life between the tides (e.g. Pfingstl, 2017). They use elaborate plastron respiration systems to withstand flooded conditions during high tide (e.g. Pfingstl & Krisper, 2014; Pugh, King, & Fordy, 1990), feed on intertidal algae, and occupy a wide range of coastal habitats, as, for example, rocky cliffs, boulder beaches, or mangrove forests (Pfingstl, 2013a). Some species are known to be island endemics, whereas others are distributed across large archipelagos and oceanic regions (Pfingstl & Schuster, 2014). In the latter case, populations from different islands or regions may show morphological diversification that is related to geographic distance between the landmasses and these divergences are supposed to indicate ongoing speciation processes due to restricted gene flow between populations (Pfingstl & Baumann, 2017; Pfingstl & Jagersbacher-Baumann, 2016). How these tiny flightless arthropods disperse between the islands is still unknown but several authors argue that drifting along ocean currents is the most likely mode of long distance transport (Coulson, Hodkinson, Webb, & Harrison, 2002; Pfingstl, 2017; Schatz, 1991). Another interesting evolutionary phenomenon, namely cryptic diversity, was also demonstrated in these intertidal mites, with species occurring on the same island, possessing almost identical appearance but occupying different ecological niches within the intertidal habitat (Pfingstl, Lienhard, & Jagersbacher-Baumann, 2014). The number of described species of the subtropical and tropical Fortuyniidae and Selenoribatidae nearly doubled in the last decade. Presently, the Fortuyniidae contain four genera (Alismobates, Circellobates, Fortuynia, and Litoribates) with 26 species, whereas the Selenoribatidae comprise eight genera (Arotrobates, Carinozetes, Psednobates, Rhizophobates, Schusteria, Selenoribates, Thalassozetes, and Thasecazetes). Despite these recent findings, knowledge about the evolution, phylogeny, and distribution of these taxa is still largely incomplete and even existing systematic classifications remain controversial. For example, the generic diagnosis of the selenoribatid Schusteria Grandjean, 1968 has repeatedly been subject to misinterpretations and erroneous taxonomic actions (Pfingstl & Schuster, 2012) leading to a blurry picture of this taxon and closely related genera. A recent molecular genetic study even questioned the monophyletic status of the family Fortuyniidae suggesting that certain fortuyniid taxa may indeed belong to the Selenoribatidae (Iseki & Karasawa, 2014). Apart from these systematic problems, the diversity of both families is largely underestimated as indicated by the recent discovery of numerous new species and genera from the Red Sea, the Eastern Pacific, the Indo-Pacific, and the Caribbean area (e.g. Pfingstl, 2015a; Pfingstl, Baumann, Lienhard, & Schatz, 2017; Pfingstl & Jagersbacher-Baumann, 2016; Pfingstl & Schatz, 2017). During an international expedition to investigate the biodiversity of interstitial and intertidal habitats of selected coastal areas of Malaysia and Thailand, littoral mites were found at various sampling sites. This material contained two new selenoribatid taxa but also three fortuyniid species known from far distant areas. Presently, nothing is known about the dispersal abilities and gene flow between the populations of these intertidal mite species showing such wide distribution areas with enormous oceanic barriers in between. Therefore, the aims of this study were (a) to document and discuss the biogeographic pattern found for oribatid mites in the Andaman Sea; (b) to compare distant populations of supposedly widespread species with morphometric and molecular genetic approaches in order to assess dispersal abilities and gene flow; (c) to describe a new genus with two species; and (d) to provide the first insight into the biodiversity of intertidal mites from this biologically interesting geographic region. 2 MATERIALS AND METHODS Samples of intertidal algae were scraped off rocks with a knife during low tide. Algae were then put in Berlese-Tullgren funnels for approx. 24 hr to extract mites. Afterward, mites were removed from the collecting vessel by hand with a brush and stored in absolute (100%) ethanol for morphological and molecular genetic investigation. Morphological terminology used in this study follows that of Grandjean, 1953 and Norton & Behan-Pelletier, 2009. Formulas for leg setation are provided in parentheses according to the sequence trochanter–femur–genu–tibia–tarsus followed by formulas for leg solenidia also given in parentheses according to the sequence genu–tibia–tarsus. 2.1 Sample locations The Andaman Sea is situated in the eastern part of the Indian Ocean and lies southeast of the Bay of Bengal for overview map see Figure S1. It extends from the Andaman and Nicobar Islands in the West to the coast of southern Myanmar in the North downward to the Thai-Malay peninsula and to the Strait of Malacca in the South. The latter is a narrow body of water between the Malay Peninsula and the Indonesian island of Sumatra (for an overview map of the region please see Supporting Information Figure S1). The climate of the Andaman region is strongly influenced by the tropical monsoons of Southeast Asia with wind systems reversing directions every year. Consequently, the climate is greatly influencing the hydrographic parameters and water movement in the area (e.g. Kiran, 2017; Wyrtki, 1961). Sampling locations were as follows: Langkawi, Malaysia: (a) Pantai Legenda; Bostrychia sp. (red intertidal algae) growing on boulder, upper eulittoral (MY_05), coordinates 6°18′42.91′′N 99°51′06.47′′E; October 24, 2016. (b) Pantai Pasir Hitam; diverse algae growing on rock, upper eulittoral (MY_07), coordinates 6°25′24.24′′N 99°47′15.81′′E; October 25, 2016. (c) Datai Bay; Bostrychia sp. growing on rock, upper eulittoral (MY_11), coordinates 6°26′01.39′′N 99°41′03.17′′E; and red filiform algae on stones, lower eulittoral (MY_12), coordinates 6°26′02.04′′N 99°40′54.51′′E; October 26, 2016. Penang, Malaysia: (d) Pantai Pasir Panjang; Bostrychia sp. growing on large rocks, upper eulittoral (MY_17), coordinates 5°18′01.61′′N 100°11′03.95′′E; October 28, 2016. Phang Nga province, Thailand: (e) Nang Thong Beach, Takua Pa district; short brown algae covering boulder; medium eulittoral (TH_06), coordinates 8°37′46.76′′N 98°14′35.95′′E; November 6, 2016. (f) Nang Thong Beach; Bostrychia sp. growing in crevice, upper eulittoral (TH_09), coordinates 8°38′07.63′′N 98°14′39.77′′E; November 8, 2016. 2.2 Genetic analyses Seventy-nine specimens of all ameronothroid mites (see Appendix) collected in Thailand and Malaysia were analyzed. Therefore, total genomic DNA was extracted from single individuals preserved in absolute ethanol. Extraction was carried out using the Chelex method (Casquet, Thebaud, & Gillespie, 2012) with the following adjustments: Whole specimens were crushed against the tube wall in microcentrifuge tubes containing 55 μl of a 10% Chelex solution (with 2 μl Proteinase K). Samples were extracted for 3–4 hr at 56°C. Three gene fragments were sequenced for this study: the mitochondrial cytochrome c oxidase subunit 1 gene (COI), the nuclear elongation factor 1 alpha gene (EF-1α), and the nuclear 18S rRNA gene (18S). A 567-bp fragment of the COI gene was amplified using the primer pairs Mite COI-2F and Mite COI-2R (Otto & Wilson, 2001), and for the 513 bp containing EF-1α gene fragment, the primer 40.71F and 52.RC (Regier & Shultz, 1997) were used. The complete 18S rRNA gene (~1.8 kb) was amplified in two overlapping fragments according to the PCR protocol of Dabert, Witalinski, Kazmierski, Olszanowski, and Dabert (2010) using the recommended primers (Skoracka & Dabert, 2010). Primer sequences are given in Supporting Information Table S4. PCR conditions for the COI gene fragment are given in Pfingstl et al. (2014) and those for the EF-1α gene fragment in Lienhard, Schäffer, Krisper, and Sturmbauer (2014). DNA purification (with the enzyme cleaner ExoSAP-IT, Affymetrix; and the Sephadex G-50 resin, GE Healthcare) and sequencing steps (using the BigDye Sequence Terminator v3.1 Cycle Sequencing Kit, Applied Biosystems) were conducted after the methods published by Schäffer, Krisper, Pfingstl, and Sturmbauer (2008). Sequencing was performed in both directions on an automated capillary sequencer (ABI PRISM 3130xl, Applied Biosystems). Alignments were generated by means of the program MEGA6 (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013). For all gene fragments, Bayesian 50% majority rule consensus trees were generated by means of MrBAYES 3.1.2 (Ronquist & Huelsenbeck, 2003) applying a MC3 simulation with 20 million generations (five chains, two independent runs, 10% burn-in, GTR + I + G model). Results were analyzed in TRACER v.1.6 (Rambaut & Drummond, 2007) to check for convergence and to ensure the stationarity of all parameters. Neighbor joining (NJ) trees were generated with MEGA6 (5000 bootstrap replicates) and maximum-likelihood (ML) analyses were carried out using RAxML (Stamatakis, 2014) applying 5000 bootstrap replicates and the GTR + gamma model. To determine the geographic correspondence with the genetic structure, TCS networks were constructed with the program PopART (Leigh & Bryant, 2015, http://popart.otago.ac.nz) applying default settings. Uncorrected p-distances were calculated in MEGA6. All sequences obtained from this study were deposited in GenBank (www.ncbi.nlm.nih.gov/genbank; accession numbers for COI: MH285595–MH285673, EF-1α: MH285674–MH285689, and 18S: MH285690–MH285696; more details are given in the Appendix). For the 18S dataset, all already published ameronothroid sequences were also integrated into the alignment. 2.3 Morphometric analyses For morphometric investigation, 81 specimens in total (not the same as used in molecular genetic analyses) were placed in lactic acid (temporary slides) and measurements were performed using a compound light microscope (Olympus BH-2) and ocular micrometre. A total of 15 continuous variables were measured in 30 specimens of A. pseudoreticulatus from three different localities on Langkawi (MY_05, MY_07, MY_11), and 15 continuous variables were taken in 51 specimens of Indopacifica pantai n. sp. from the island of Penang (MY_17) in Malaysia and from Phang Nga province (TH_09) in Thailand (Figures 1a,b). Figure 1Open in figure viewerPowerPoint Graphic illustration of measured continuous variables. (a) Alismobates pseudoreticulatus. (b) Indopacifica pantai n. sp. Dorsal aspect: bl: body length; dPtI: distance between pedotecta I, db: distance between bothridia; ll: lenticulus length; nwc1: notogastral width on level of seta c1; nwda: notogastral width on level of seta da; nwdm: notogastral width on level of seta dm. Ventral aspect: cl: camerostome length; cw: camerostome width; efw: epimeral fovea width; dcg: distance between camerostome and genital orifice; dac3: distance between acetabula 3; gl: genital orifice length; gw: genital orifice width; al: anal opening length; aw: anal opening width For univariate statistics minimum, maximum, mean, standard deviation, and coefficient of variation (cv) were calculated. These numbers were calculated to assess variation within but also between populations. Mann–Whitney U test was used for comparing the means of variables for pairwise comparisons in order to clarify if single variables differ significantly between two populations. Principal component analysis (PCA) was performed on log10-transformed raw and size-corrected data using a variance–covariance matrix. No rotation was applied to the multivariate data. Size correction was done by dividing each variable through the geometric mean of the respective specimen (e.g. Jagersbacher-Baumann, 2014; Pfingstl & Jagersbacher-Baumann, 2016). All analyses were performed with PAST 3.11 (Hammer, Harper, & Ryan, 2001). 2.4 Drawings and photographs For microscopic investigation in transmitted light, preserved animals were embedded in Berlese mountant. Drawings were made with an Olympus BH-2 Microscope equipped with a drawing attachment. These drawings were first scanned, then processed, and digitized with the free and open-source vector graphics editor Inkscape (https://inkscape.org). For photographic documentation, specimens were air-dried and photographed with a Keyence VHX-5000 digital microscope. 3 RESULTS 3.1 Molecular genetics Bayesian inference gene trees, based on the mitochondrial (COI) as well as on the nuclear marker (EF-1α), revealed six highly divergent, well supported (all posterior probability values 100) and monophyletic lineages referring to six distinct species (Figure 2). Because of the higher statistical support, BI topologies are shown herein, although NJ and ML analyses resulted in the exact same clades. Within the Selenoribatidae, two clades are present harboring two new species of the genus Indopacifica n. gen., and within the Fortuyniidae, one lineage refers to A. pseudoreticulatus and three further clades represent the genus Fortuynia (F. smiti, F. sp., and F. longiseta). The distinctness of the species is demonstrated by the clear gap between mean intra- and interspecific p-distances of both markers (3.0 vs. 14.6% COI; 0.9 vs. 6.5% EF-1α; Table 1). For the COI gene fragment, the highest intraspecific p-distance amounted to 6.4% (I. pantai n. sp.) compared to the lowest interspecific distance of 14.0%. For the EF-1α gene fragment, the highest intraspecific p-distance reached 1.6% (I. pantai n. sp.) compared to the lowest interspecific distance of 6.3% between two fortuyniid species. Alismobates pseudoreticulatus as well as both Indopacifica species are distributed in Malaysia and Thailand, but haplotypes of A. pseudoreticulatus are clearly associated with their geographic distribution (Figures 3a,b and 11a,b). Indopacifica pantai n. sp. as well as I. parva n. sp., on the other hand, show no clear geographic structure (Figures 3a,b and 11a). Data for fortuyniid species were less comprehensive; therefore, only one haplotype for Fortuynia sp. and F. smiti and three different haplotypes for F. longiseta were detected. To resolve deeper splits in the phylogeny and to compare the phylogenetic position of the new genus and species to already published data, 18S sequences were gained and compared (Figure 4). These sequences correspond to known ones, and no conflicting positions were detected. The monophyly of the new genus Indopacifica is strongly supported, and the two species are clearly distinct from each other. Figure 2Open in figure viewerPowerPoint Bayesian inference trees based on COI (left) and EF-1α (right) sequences. Species are grouped in colored boxes with corresponding micrographs of adult individuals. Posterior probabilities (>95%) are shown above branches Table 1. Intra- and interspecific mean p-distances given for the COI (lower-left) and the EF-1α (upper-right) gene fragment in percent Fortuynia smiti (2) Fortuynia longiseta (2) Fortuynia sp. (2) Alismobates pseudoreticulatus (4) Indopacifica pantai (4) Indopacifica parva (2) Fortuynia smiti (9) 0.0/0.4 8.4 7.1 15.0 14.8 14.5 Fortuynia longiseta (4) 15.4 0.4/0.4 6.5 15.4 14.9 14.9 Fortuynia sp. (7) 15.9 15.9 0.0/0.0 15.8 15.6 14.9 Alismobates pseudoreticulatus (25) 15.5 16.1 14.6 0.5/0.3 14.8 13.8 Indopacifica pantai (30) 17.5 16.1 18.7 15.5 3.0/0.9 9.1 Indopacifica parva (4) 16.0 18.7 16.8 14.6 17.0 1.6/0.4 Note The number of investigated specimens is given in parenthesis. Figure 3Open in figure viewerPowerPoint TCS haplotype networks based on COI sequences including three ameronothroid genera, namely Alismobates (a), Fortuynia (b), and Indopacifica (c). Each circle corresponds to one haplotype, and its size is proportional to its frequency, the number of mutations are indicated as hatch marks. Small black circles represent intermediate haplotypes not present in the dataset. Colors refer to different locations in Malaysia and Thailand and correspond to those in Figure 11a,b Figure 4Open in figure viewerPowerPoint Bayesian inference tree based on 18S sequences. Posterior probabilities (>80) are shown on the nodes. Sequences obtained from GenBank are marked by an asterisk (*) 3.2 Morphometry 3.2.1 Univariate statistics of Alismobates pseudoreticulatus populations The A. pseudoreticulatus populations from Langkawi differed highly significantly (p < 0.001) in body length (bl) and posterior notogastral width (nwdp) when compared by Mann–Whitney U test (Table 2). The specimens from the north shore (MY_07) were slightly longer and broader than the specimens from the south shore (MY_05). The variability as indicated by the coefficient of variation (cv) was moderate in all populations with values hardly exceeding 0.05. The most variable characters were the lenticulus length (ll) and the posterior notogastral width (nwdp). Table 2. Univariate statistics for Alismobates pseudoreticulatus populations from three different locations (MY_05, MY_07, and MY_11) on the island of Langkawi Variables Alismobates pseudoreticulatus MY_05 (N = 15) MY_07 (N = 14) MY_11 (N = 1) cv MWU bl 295–325 (309 ± 7.69) 306–332 (319 ± 6.76) 302 0.03 * dPtI 135–147 (143 ± 2.89) 140–148 (143 ± 2.39) 142 0.02 ** db 77–86 (91 ± 3.05) 77–86 (82 ± 2.10) 83 0.03 ** ll 55–71 (64 ± 4.52) 55–71 (62 ± 5.20) 55 0.08 ** nw da 197–231 (211 ± 9.09) 206–231 (216 ± 7.64) 206 0.04 ** nw dm 209–234 (221 ± 6.87) 219–240 (229 ± 5.55) 225 0.03 ** nw dp 169–200 (180 ± 7.76) 179–212 (193 ± 8.77) 200 0.06 * cl 77–86 (81 ± 3.18) 77–86 (80 ± 2.59) 80 0.04 ** cw 68–80 (74 ± 3.18) 69–77 (73 ± 1.91) 71 0.04 ** dcg 68–80 (73 ± 3.25) 71–80 (76 ± 3.32 74 0.05 ** dac3 123–132 (127 ± 2.66) 123–132 (128 ± 2.27) 129 0.02 ** gl 43–52 (49 ± 2.36) 46–55 (49 ± 2.82) 46 0.05 ** gw 55–65 (59 ± 2.53) 59–66 (61 ± 2.01) 59 0.04 ** al 65–71 (67 ± 2.07) 65–75 (70 ± 2.42) 68 0.04 ** aw 52–62 (57 ± 2.61) 55–62 (58 ± 2.16) 59 0.04 ** Notes. Minimum–maximum (mean ± standard deviation) of each measured variable given in μm; cv—coefficient of variation, values higher than 0.5 are given in bold. MWU—Mann–Whitney U test (comparing the medians of the populations). Abbreviations for variables are explained in caption of Figure 1. *p < 0.001, **p > 0.001; single specimen from MY_11 not included in this test. 3.2.2 Multivariate analysis of Alismobates pseudoreticulatus Principal component analysis on both raw and size-corrected data showed a slight misalignment of the populations indicating a trend toward morphological divergence (Figure 5a). In raw data, PC1 accounted for 37.14%, PC 2 for 27.04%, and PC3 for 9.82% of total variation and loadings higher than 0.5 are given for the lenticulus length (ll) and for the posterior notogastral width (nwdp) (Supporting Information Table S1). Similar results were shown for the size-corrected data; PC1 is responsible for 49.51%, PC2 for 22.53%, and PC3 for 7.39% of total variation, whereas the variables bl and nwdp showed loadings with remarkably high values (>0.5). Figure 5Open in figure viewerPowerPoint Graphs showing results of principal component analyses performed with raw data and size-corrected data of two different species from different locations in the Andaman Sea and the Strait of Malacca. (a) Alismobates pseudoreticulatus. (b) Indopacifica pantai n. sp. Different populations represented by different colors. Open symbols refer to males, and filled symbols refer to female specimens. Codes (e.g. MY_05) refer to sample locations 3.2.3 Univariate statistics of Indopacifica pantai n. sp. populations Indopacifica pantai n. sp. populations from Malaysia and Thailand only differed significantly (p < 0.001) in the distance between bothridia (db) as indicated by Mann–Whitney U test (Table 3). The bothridia of the Thai specimens (TH_09) were slightly farther apart than in the Malaysian specimens (MY_17). The variability was basically low, only the epimeral fovea (efw) and the size of genital orifice (gl, gw) show higher coefficients of variation with 0.13 and 0.09, respectively. Except efw, these variables are related to a moderate sexual dimorphism. Table 3. Univariate statistics for Indopacifica pantai n. sp. populations from two different locations in the Andaman Sea and the Strait of Malacca Variables Indopacifica pantai MY_17/Penang (N = 23) TH_09/Phang Nga (N = 28) cv MWU bl 319–356 (333 ± 9.84) 319–350 (332 ± 8.10) 0.03 ** dPtI 139–154 (147 ± 3.88) 142–154 (148 ± 2.71) 0.02 ** db 52–68 (63 ± 3.44) 61–71 (66 ± 2.17) 0.05 * nw da 175–206 (192 ± 7.94) 185–206 (194 ± 5.89) 0.04 ** nw dm 191–215 (205 ± 5.98) 197–219 (208 ± 5.69) 0.03 ** nw dp 163–185 (175 ± 6.41) 169–194 (181 ± 6.33) 0.04 ** cl 83–92 (90 ± 2.42) 62–94 (90 ± 5.68) 0.05 ** cw 62–68 (65 ± 1.79) 62–68 (65 ± 1.50) 0.02 ** efw 19–34 (28 ± 3.74) 22–31 (26 ± 2.93) 0.13 ** dcg 71–83 (76 ± 3.75) 74–86 (80 ± 3.35) 0.05 ** dac3 111–123 (116 ± 3.23) 108–120 (117 ± 2.64) 0.03 ** gl 40–55 (48 ± 4.72) 42–55 (48 ± 3.91) 0.09 ** gw 46–59 (53 ± 5.17) 49–62 (54 ± 4.86) 0.09 ** al 71–77 (74 ± 2.08) 69–77 (73 ± 1.93) 0.03 ** aw 52–59 (55 ± 2.62) 52–62 (57 ± 2.53) 0.05 ** Notes. Minimum–maximum (mean ± standard deviation) of each measured variable given in μm; cv—coefficient of variation, values higher than 0.5 are given in bold. MWU—Mann–Whitney U test (comparing the medians of the two populations). Abbreviations for variables are explained in caption of Figure 1. *p < 0.001, **p > 0.001. 3.2.4 Multivariate analysis of I. pantai n. sp The PCA on raw and size-corrected data resulted in mainly overlapping clusters between the populations from Mala