Altrichthys alelia, a new brooding damselfish (Teleostei, Perciformes, Pomacentridae) from Busuanga Island, Philippines

Abstract A new species of damselfish, Altrichthys alelia sp. n. is described from specimens collected in shallow water (1–8m depth) off Busuanga Island, Palawan Province, Philippines. It differs from the other two species in the genus, A. curatus and A. azurelineatus, in various features including having golden upper body lacking dark edges of dorsal and caudal fins, higher modal number of tubed lateral line scales, as well as differences in two mitochondrial markers, one nuclear marker, and RAD markers.


Introduction
The damselfish genus Altrichthys Allen, 1999 includes two species, the azure damselfish, Altrichthys azurelineatus (Fowler & Bean, 1928), and the guardian damselfish, Altrichthys curatus Allen, 1999, that occur on shallow coral reefs in the Calamian Archipelago, northern Palawan Province, Philippines (Allen 1999, Bernardi 2011, Bernardi et al. 2017. While conducting exploratory dives in the less-studied area of northern Busuanga Island, in the region of San José, we observed and collected Altrichthys individuals that after closer examination and laboratory work showed unique morphological and genetic characters that distinguish them from previously described Altrichthys species. We can therefore confirm the presence of a third Altrichthys species that we hereby describe as the new species Altrichthys alelia (Figure 1).

Methods and materials
Specimens were collected with hand nets while free or scuba diving in less than 8m depth. Counts and measurements follow Allen (1999). Measurements were made days to a few weeks after collection and were taken to the nearest 0.1 mm with digital calipers. Collection abbreviations are as follows: PNM = National Musem of the Philippines, CAS = California Academy of Sciences.
Fin clip tissue samples were stored in 95% ethanol and DNA was extracted using DNeasy Blood & Tissue kits (Qiagen) according to the manufacturer's protocol. DNA was extracted from two Altrichthys alelia individuals (juvenile individuals 6 and 8, in CAS 241439), and from five individuals of each sister species, A. azurelineatus and A. curatus collected at Uson Island and Sangat Island (Figure 2). Extractions were PCR amplified for two mitochondrial (control region and cytochrome oxidase 1) and one nuclear marker (RAG2) following published protocols (Bernardi 2011) (Genbank accession numbers KY963970-KY963994; KY969587, KY969588). Phylogenetic reconstructions were done based on the Neighbor-Joining method  generated in R (R Core Team 2013) with the ape package (Paradis et al. 2004) using Kimura-2 parameter substitution models, and a Maximum Likelihood method implemented in GARLI (Zwickl 2006). Node support was obtained using 1000 bootstrap replicates and retaining values that support nodes in more than 50% of the bootstrap replicates.

Alelia's damselfish
Diagnosis and description. A species of Altrichthys distinguished by the following combination of characters: dorsal rays XIV, 13-14; anal rays II, 15, tubed lateral line scales 14-15 (Table 1); preorbital and sensory pores small and numerous, usually more than 30, adult coloration in life pale green on upper half grading to white on lower part; iris silvery; pale yellow to gold outer margin of dorsal and upper and lower edges of caudal fin. Fins mainly white to translucent. Juveniles up to 16mm in length are mostly white with a prominent yellow stripe along the lateral line ( Figure 3). Adults are generally of the same size as other Altrichthys adults, approximately 70-80mm TL. Altrichthys alelia differs from A. curatus by having long filaments at the trailing edges of the dorsal and caudal fins, and from A. azurelineatus by lacking any black lining of the outer edges of dorsal and caudal fins. These black margins are represented by yellow/gold margins in A. alelia (Figure 4). Pored lateral In addition, Sanger sequencing results show that Altrichthys alelia individuals form a group most closely related to, but distinct from, A. azurelineatus ( Figure 5). Altrichthys alelia sequences differed from their closest relative A. azurelineatus by one fixed difference at the nuclear locus RAG2. For mitochondrial markers, Altrichthys alelia sequences differed from A. azurelineatus by 13 and 15 fixed mutations for cytochrome oxidase I and control region markers, respectively, thus corresponding to a Kimura-2 sequence divergence of 2.3% and 5.9% respectively. These divergences are consistent with values obtained in other sister species of fish (Ward et al. 2005). RAD DNA sequencing results were also consistent with A. alelia and A. azurelineatus being distinct species and more closely related to each other than either are to A. curatus (Table 2). Indeed, RAD sequencing generated 8383 variable SNPs for the 5 sequenced individuals (2 A. alelia, 1 A. azurelineatus, 2 A. curatus). Of those 8383 SNPs, 1584.5 (18.9%) showed differences between A. alelia and A. azurelineatus; while 7224.5 (86.2%) and 6495 (76.4%) showed differences between A. curatus and A. alelia and between A. curatus and A. azurelineatus, respectively (Table 2).

Habitat. Collected off live and extensive thickets of corals mostly Porites cylindrica.
Etymology. The name Altrichthys alelia derives from the combined first names of Alessio Bernardi and Amalia Bernardi, who greatly helped during field-work on Altrichthys.
Common name. We suggest Alelia's damselfish as a literal translation of the scientific name.

Discussion
Species of the genus Altrichthys together with their close relative Acanthochromis polyacanthus are unusual as they brood their young (Allen 1999, Bernardi et al. 2017, whereas most coral reef fishes have a pelagic larval stage (Leis 1991). This behavior is clearly visible on the reef, by the presence of pairs of adult fish aggressively defending a cloud of fry that remain around the parents. An identical behavior was observed in A. alelia, where fry and adult pairs were associated, and similarly to the other Altrichthys species, Porites cylindrica is used as a nesting substrate (Bernardi et al. 2017). While Altrichthys azurelineatus and A. curatus co-occur at most locations in southern Busuanga, we did not observe any other Altrichthys species occurring in the vicinity of A. alelia.
Altrichthys alelia may have been observed previously by other scientists, but remained unnoticed. The original description of Chromis azurelineatus (Fowler & Bean, 1928), later re-described as Altrichthys azurelineatus (Allen, 1999), was based on fish collected during the Albatross expedition of 1908-1909. Three collections were made in December 1908 (Smith and Williams 1999). The collection that yielded the type specimen was made in Uson Island, using dynamite. The authors spent several days on Uson Island, and observed hundreds of A. azurelineatus there but never saw A. alelia in that locality. That specimen indeed looks like a genuine A. azurelineatus with its distinctive dark caudal fin margins (USNM, 89957, Figure 6, top right panel). The other collections of the Albatross were at Tara island, which faces the north-eastern portion Busuanga Island, a region close to the type locality of A. alelia (Figure 2). Fish collected at Tara island have "dorsal spines bright orange, color carried somewhat into membranes in another portion of the fin" (Fowler and Bean 1928). The geographic location and description are consistent with A. alelia, however the lack of diagnostic morphological and meristic characters precludes final determination if this were the case. Nevertheless, specimens from these collections lack the characteristic black margins on the caudal fin that is found in A. azurelineatus (USNM, 96398, and USNM, 96425, Figure 6, bottom left and bottm right panels). The drawing of A. azurelineatus in the original description by Fowler and Bean (1928), and later redrawn in color (Allen 1991), does not show the dark margins either (Figure 6, top left panel), as it may have been a compilation of all the various specimens collected (i.e. a combination of A. azurelineatus and A. alelia individuals). As such, that drawing is more similar to A. alelia. Finally, the picture taken on Busuanga island labeled as A. curatus in Figure 2 of Allen (1999), looks precisely like A. alelia. Due to the peculiarities of this fish, Allen (1999) suggested that "additional observations are needed", which is what we are presenting here. discussing our data and invaluable information on Altrichthys. We are very grateful to Sandra J. Raredon for taking pictures of Altrichthys specimens at the Smithsonian collection, and to Don Dumale (National Museum of the Philippines) and David Catania (California Academy of Science) for providing Holotype and Paratype numbers. This work was funded by a National Geographic Grant and a UCSC Committee on Research (COR) grant to GB. Smith DG, Williams JT (1999)