Systematics of Damselfishes Author Tang, Kevin L. Author Stiassny, Melanie L. J. Author Mayden, Richard L. Author DeSalle, Robert text Ichthyology & Herpetology 2021 2021-05-05 109 1 258 318 http://dx.doi.org/10.1643/i2020105 journal article 53279 10.1643/i2020105 cf572f6b-8843-4383-85ce-ac9ea8515e87 2766-1520 7846738 Tribe Amphiprionini . — Of all the clades within the Pomacentrinae , this group is the most distinctive as it hosts all of the anemonefishes. Their monophyly (100% bootstrap) is not surprising given their obligate symbiotic relationship with sea anemones, a life history trait that is unique across fish diversity (Collingwood, 1868; de Crespigny, 1869; Verwey, 1930 ; Gudger, 1946 ; Mariscal, 1970 ; Allen, 1972; Fautin, 1991 ; Fautin and Allen, 1997 ). The symbiosis has been credited with triggering the rapid diversification ( Litsios et al., 2012a ; Marcionetti et al., 2019 ) that gave rise to 30 currently recognized species found across the Indo-West Pacific. Anemonefishes share morphological characters that further distinguish them from other damselfishes, including serration of the infraorbital and opercular series, reduced number of dorsal spines (VIII–XI vs. XII or more), and small scales (Allen, 1972; Fitzpatrick, 1992 ; Tang, 2002 ). As a result, this clade has been one of the most strongly supported within the family in past phylogenies (e.g., Quenouille et al., 2004 ; Cooper et al., 2009; Litsios et al., 2012a ). Although evidence for their monophyly is robust, the exact number of species is uncertain. There are currently either 28 or 30 recognized in this tribe, depending on the status of A. leucokranos and A. thiellei , two putative species of possible hybrid origin ( Fautin and Allen, 1997 ; Ollerton et al., 2007 ; see below). The anemonefish relationships reported herein share some similarities with aspects of earlier works (e.g., Santini and Polacco, 2006 ; Litsios et al., 2012 a , 2014; Litsios and Salamin, 2014 ; O’Donnell, 2014 ; Dhaneesh et al., 2015; Rolland et al., 2018 ; Thongtam na Ayudhaya et al., 2019 ; Nguyen et al., 2020 ). A monophyletic group (100% bootstrap) composed of the two clownfishes ( A. ocellaris and A. percula ) and Premnas is sister to all other amphiprionins. Two morphological characters have been hypothesized to unite these three species: distinct notch at dorsal-fin junction (spinous vs. soft sections) and naked occipital region (Allen, 1972; Fitzpatrick, 1992; Tang, 2002 ). Concerning the first trait, Salis et al. (2018) discovered a correlation between the extent of dorsal-fin indentation and the number of vertical white bars ( sensu Barlow, 1972 ; Hensley and Randall, 1983 ). These three species display the greatest number of bars (3) among anemonefishes, a condition shared with several other species (e.g., A. clarkii , A. latezonatus , A. tricinctus ). Amphiprion latezonatus is sister to a clade of the remaining anemonefishes (100% bootstrap). It is in the crown group where the relationships shown herein begin to diverge from other phylogenies, which also disagree with each other in some instances. Part of the discordance could be the result of gene choice because there is evidence of incongruence between the mitochondrial and nuclear data in these fishes ( Litsios and Salamin, 2014 ). The instability in this part of the tree might also stem from a lack of sufficient phylogenetically informative variation due to rapid diversification. The genus appears to have undergone a recent radiation where the bulk of its diversity has arisen within the last 5–10 million years ( Santini et al., 2009 ; Cowman and Bellwood, 2011, 2013; Litsios et al., 2012a ; Frédérich et al., 2013 ; Lobato et al., 2014 ; DiBattista et al., 2016; Rabosky et al., 2018 ; Rolland et al., 2018 ). Several areas of agreement do emerge among different studies: a monophyletic A. polymnus and A. sebae ; a monophyletic skunk clade consisting of A. akallopisos , A. pacificus , A. perideraion , and A. sandaracinos ; an ‘‘ ephippium complex’’ sensu Allen (1980) , minus A. mccullochi , consisting of A. barberi , A. ephippium ( type species), A. frenatus , A. melanopus , and A. rubrocinctus ; an Indian Ocean clade consisting of A. allardi , A. bicinctus , A. chagosensis , A. chrysogaster , A. latifasciatus , A. nigripes , and A. omanensis . The latter group appears to be a recent invasion into the Indian Ocean followed by rapid diversification (Cowman and Bellwood, 2013: fig. S4; Litsios et al., 2014). This is where our tree diverges most dramatically from the consensus, as we recovered A. polymnus and A. sebae nested deep within the Indian Ocean clade, which has not been reported in other studies except Nguyen et al. (2020 : fig. 2) who found a similar relationship for A. polymnus ( A. sebae was recovered with A. clarkii therein). Allen (1972) treated Premnas as a subgenus of Amphiprion , stating that the two most prominent characters used to differentiate Premnas , prominent infraorbital spine and high number of transverse scale rows, were of ‘‘relatively minor phylogenetic importance.’’ Allen (1975a, 1975b) reversed this decision. However, early molecular studies ( Tang, 2001 ; Quenouille et al., 2004 ; Santini and Polacco, 2006 ) resolved the monotypic Premnas within Amphiprion , prompting some (e.g., Quenouille et al., 2004 ; Nelson , 2006 ; Santini and Polacco, 2006 ) to return Premnas to the synonymy of Amphiprion . Although some phylogenies have resolved Premnas as the sister group to Amphiprion (e.g., Mirande, 2016 ; Thongtam na Ayudhaya et al., 2017 ), a multitude of other studies (Cooper et al., 2009; Cowman and Bellwood, 2011; Litsios et al., 2012a , 2012 b , 2014; Frédérich et al., 2013 ; Rabosky et al., 2013 , 2018 ; Litsios and Salamin, 2014 ; Lobato et al., 2014 ; Dhaneesh et al., 2015; Li et al., 2015 ; DiBattista et al., 2016; Gaboriau et al., 2018 ; Rolland et al., 2018 ; Delrieu-Trottin et al., 2019; Marcionetti et al., 2019 ; Thongtam na Ayudhaya et al., 2019 ) have recovered Premnas biaculeatus within Amphiprion , as the sister group of the clownfishes ( A. ocellaris þ A. percula ). Nonetheless, workers have continued to recognize Premnas as a distinct genus despite its phylogenetic position. Based on the relationships presented herein, as well as the overwhelming consensus from past studies, we treat Premnas as a junior synonym of Amphiprion . Both genera are masculine in gender, so the species name remains unchanged in the new combination as Amphiprion biaculeatus . Allen (1975a, 1975b) recognized four subgenera within Amphiprion : Actinicola , Amphiprion , Paramphiprion , and Phalerebus . Allen (1975b, 1980, 1991) further subdivided the subgenus Amphiprion into an ephippium -complex ( A. ephippium , A. frenatus , A. mccullochi , A. melanopus , A. rubrocinctus ) and a clarkii -complex (remaining species of the subgenus). Our study and many others ( Koh et al., 2006 ; Santini and Polacco, 2006 ; Timm et al., 2008; Cooper et al., 2009; Cowman and Bellwood, 2011; Litsios et al., 2012a , 2012 b , 2014; Frédérich et al., 2013 ; Rabosky et al., 2013 , 2018 ; Litsios and Salamin, 2014 ; O’Donnell, 2014 ; Dhaneesh et al., 2015; Li et al., 2015 ; DiBattista et al., 2016; Mirande, 2016 ; Thongtam na Ayudhaya et al., 2017 , 2019 ; Gaboriau et al., 2018 ; Rolland et al., 2018 ; Delrieu-Trottin et al., 2019; Nguyen et al., 2020 ) have provided ample evidence for the monophyly of Actinicola , which encompasses the two clownfish species, A. ocellaris and A. percula . As a subgenus of Amphiprion , Premnas is sister to Actinicola . Paramphiprion is not monophyletic; A. polymnus and A. sebae are sister species but A. latezonatus is never recovered with them. Instead, A. latezonatus is often found as the sister group of all Amphiprion excluding the subgenera Actinicola and Premnas ( Santini and Polacco, 2006 ; Cowman and Bellwood, 2011; Frédérich et al., 2013 ; Litsios and Salamin, 2014 ; Litsios et al., 2014; O’Donnell, 2014 ; DiBattista et al., 2016; Mirande, 2016 ; Gaboriau et al., 2018 ; Rolland et al., 2018 ; Delrieu-Trottin et al., 2019; Nguyen et al., 2020 ). Phalerebus is not monophyletic either, with A. nigripes recovered apart from the skunk anemonefishes ( A. akallopisos , A. pacificus , A. perideraion , A. sandaracinos ), which do form a clade (100% bootstrap). A monophyletic Phalerebus without A. nigripes is also seen in many other studies (e.g., Santini and Polacco, 2006 ; Timm et al., 2008; Steinke et al., 2009 ; Cowman and Bellwood, 2011; Litsios et al., 2012 a , 2014; Litsios and Salamin, 2014 ; O’Donnell, 2014 ; Dhaneesh et al., 2015; DiBattista et al., 2016; Mirande, 2016 ; Thongtam na Ayudhaya et al., 2017 ; Gaboriau et al., 2018 ; Rolland et al., 2018 ; Delrieu-Trottin et al., 2019; Nguyen et al., 2020 ). These four species share similarities in color pattern (dorsal white stripe), body shape (slender body where body depth 2.1 in SL; rounded caudal fin), and preference for host species of the genus Heteractis (Allen, 1972, 1991; Fautin and Allen, 1997 ; Timm et al., 2008; Allen et al., 2010a). The subgenus Amphiprion , which includes the remaining anemonefish species, is rendered polyphyletic by the phylogenetic positions of putative Paramphiprion and Phalerebus . The ephippium complex ( sensu Allen, 1980 ), which is composed of species with a single bar or no bars ( A. barberi , A. ephippium , A. frenatus , A. mccullochi , A. melanopus , and A. rubrocinctus ), would be monophyletic if A. mccullochi is excluded. Other species with one or fewer bars are also found outside of this clade (e.g., A. nigripes , A. omanensis , and A. perideraion ). The subgeneric names have seen sporadic use (e.g., Elliott et al., 1999 ; Tang, 2001 ; Santini and Polacco, 2006 ; Timm et al., 2008; Steinke et al., 2009 ; Dhaneesh et al., 2015; Li et al., 2015 ; Hu et al., 2016 ; Nguyen et al., 2020 ), and any future recognition would require revision of their limits. Species boundaries for several anemonefishes may be in flux and require closer inspection. Strong population substructure has been reported in both A. ocellaris and A. percula ( Timm and Kochzius, 2008 ; Timm et al., 2008, 2012). Timm et al. (2008) detected possible cryptic diversity and hybridization within what they called the ‘‘ A. ocellaris / A. percula species complex.’’ Thongtam na Ayudhaya et al. (2017) found evidence of undescribed species diversity in not only those two species, but also in their close relative, A. biaculeatus (as P. biaculeatus ), as well as in A. bicinctus and A. clarkii . Litsios et al. (2014) suggested that A. clarkii is a complex of previously undetected species. That would not be surprising given its enormous range, extending from the Persian Gulf to the western Pacific Ocean, and highly variable coloration (Allen, 1972, 1991). Rolland et al. (2018 : fig. 1b) also illustrated a divergence within A. clarkii . Litsios et al. (2014) resolved A. chrysopterus in two separate lineages: one with individuals from Fiji and Moorea sister to a large Amphiprion clade vs. one with individuals from the Solomon Islands sister to A. akindynos þ A. mccullochi . However, Litsios and Salamin (2014) found all three populations of A. chrysopterus together in the same clade. That result was recovered in both their mitochondrial and nuclear phylogenies, though the position of A. chrysopterus differed between the two topologies ( Litsios and Salamin, 2014 : fig. 1). With access to the same samples, Rolland et al. (2018) only included A. chrysopterus from the Solomon Islands . Hubert et al. (2017 : fig. S1) also found two different lineages of A. chrysopterus , one from Moorea and the other from New Caledonia , mirroring the Fiji þ Moorea vs. Solomon Islands split observed by Litsios et al. (2014). Hybridization may have played a large role in the evolutionary history of Amphiprion (Timm et al., 2008; van der Meer et al., 2012 ; Litsios and Salamin, 2014 ; Litsios et al., 2014). Steinke et al. (2009) noted that there is ‘‘little, if any, barcode divergence’’ among the skunk anemonefishes ( A. pacificus not examined). They ruled out incomplete lineage sorting as the reason for low interspecific variation in COI, instead positing hybridization as a more likely explanation because of the widespread sympatry of these species. Based on microsatellite and mitochondrial data, van der Meer et al. (2012) suggested that A. akindynos and A. mccullochi have a history of hybridization. They also conceded that another possible explanation for their results was that A. akindynos and A. mccullochi are color morphs of a single species; they noted that further investigation would be necessary. Recent genetic studies ( Litsios and Salamin, 2014 ; Gainsford et al., 2015 ; He et al., 2018 ) have confirmed the long-standing suspicion that Amphiprion leucokranos is a natural hybrid of A. chrysopterus and A. sandaracinos (Carlson, 1996; Fautin and Allen, 1997 ; Ollerton et al., 2007 ), where A. chrysopterus , as the larger of the two species, is always the maternal parent. Three of the four sequences representing A. leucokranos in this study, all mined from GenBank (Supplemental Table 1 ; see Data Accessibility), are of mitochondrial origin. Considering the nature of its hybrid parentage, resolving it as the sister of A. chrysopterus is not surprising and matches the results shown in Litsios and Salamin (2014 : fig. 3). Because of its putative hybrid origin, A. leucokranos often is not considered a valid species (e.g., Ollerton et al., 2007 ; Litsios and Salamin, 2014 ; Litsios et al., 2014). Santini and Polacco (2006) regarded it as valid because they observed A. leucokranos forming breeding pairs with each other and to the exclusion of either parental species. However, Gainsford et al. (2015) demonstrated that, in one locality where A. leucokranos hybrids occur, there are instances of extensive hybridization and backcrossing among all three populations. Resolving the status of A. leucokranos requires considerably more investigation. The issues surrounding A. leucokranos also apply to A. thiellei , another possible hybrid believed to be produced by the same two parental species ( Fautin and Allen, 1997 ; Ollerton et al., 2007 ). If hybrids of A. chrysopterus and A. sandaracinos are recognized as a distinct species and if the hybrid origins of A. leucokranos Allen, 1973a and A. thiellei Burgess, 1981 arise from the same circumstances, then A. thiellei would need to be placed in the synonymy of A. leucokranos .