Reply to Hendawitharana et al. Comment on “Arulananthan et al. The Status of the Coral Reefs of the Jaffna Peninsula (Northern Sri Lanka), with 36 Coral Species New to Sri Lanka Confirmed by DNA Bar-Coding. Oceans 2021, 2, 509–529”

We appreciate the comments made by Hendawitharana et al. (2023) on our paper reporting on the corals of Northern Sri Lanka, including records of 38 coral species new to Sri Lanka, and genetic investigations of eight of these [1]. We agree that the identification of corals up to the species level is challenging, especially in some genera, notably Acropora, and understand the concerns raised on the use of different methods to identify coral species may lead to varying conclusions. In our study, species were primarily identified using morphological characteristics, and molecular methods only being used to check on some doubtful species. In any case, it was not completely unexpected to find species previously unreported in our study area in Northern Sri Lanka. The reefs of Northern Sri Lanka have been studied to a very small degree due to historical reasons and are located closer to the centers of coral biodiversity in the Western and Eastern Indian Oceans.

To discuss our methods further, we should emphasize that the intention was to use DNA methodology only on those species whose identity was otherwise in doubt. In regard to morphological identification, we relied principally on underwater photography to identify the species concerned. We believe that this method should be preferred where possible when working within marine protected areas or with species that appear to be scarce. However, in all cases, we took photographs from different angles and three different distances (macro, close-up, and wider view), in a way not possible before the latest generation of cameras became available. To enhance the accuracy of our identification, more than one author independently checked the identification of the sample corals by referring to both the most widely used coral reference books—The Corals of the World [2] and the World Register of Marine Species (WoRMS) [3]. In unclear cases, while one author read the description from the guide, a second simultaneously checked the macro morphological features of the coral. In some cases, the advice of other coral experts was sought.

In regard to the molecular work, we used the most common method of barcoding [4,5,6,7,8]. However, we agree that it would increase the accuracy and robustness of species identification if multiple markers, including mitochondrial COI gene, 16S rRNA, and nuclear ribosomal internal transcribed spacer (ITS) regions can be used together [8,9,10,11,12,13,14,15,16,17]. In practice, the results of our barcoding matched the conclusions reached using traditional morphology-based identification. We would emphasize that all DNA sequence chromatograms were assembled and edited using Unipro UGENE 1.29.0, and then, sequences were aligned using ClustalW [18]. The aligned outputs were queried to identify the species, and the reference sequences of various coral species were retrieved from the National Center for Biotechnology Information (NCBI) [19,20,21,22], GenBank, and Barcode of Life Data System (BOLD) [23] by using the Basic Local Alignment Search Tool (BLAST). Those sequences corresponding to the genotype were analyzed for homologies with sequences in the NCBI database by performing BLAST and BOLD workbench. From the BLAST analysis, all samples provided a greater than 99.5% similarity. Nevertheless, we did discuss the limitations of this method in the Discussion Section of our paper [1].

In most cases, Veron’s [1] “keys for genera and species” proved satisfactory for the robust identification of the corals observed. Identification of seven samples proved more challenging; these were Acropora aspera (COJP001), Acroposra digitifera (COJP003), and two specimens from two different locations representing Acropora gemmifera (COJP002, COJP004), Acropora hyacinthus (COJP006), Echinopora gemmacea (COJP007), and Montipora flabellate (COJP009), which was present only in small colony form. Even though we accept that the COI is not definitive for Acropora species, the best fitting morphological descriptions from Corals of the World [2], and we have chosen the best fitting BLASTn output to conclude the identification. It should also be noted that even though, unsurprisingly, the COI sequences of COJP001, COJP002, and COJP006 did not discriminate between them, the indicated identity reinforced the identification previously made from the study of the sample’s morphological parameters, including corallite structure (axial, radial, exserted radial), as well as colony form.

More generally, we are aware that the conventional classification of scleractinian corals based on their morphology alone has been called into question based on both molecular and small-scale morphological data, resulting in frequent revisions to their classification at all levels [8,13,14,15,24,25,26]. Advances in molecular analysis have revealed clearer inconsistencies between previously accepted coral taxonomy and their apparent evolutionary past. Further, the term “accepted” may also be used to gloss over significant differences in name games with corals. For example, Acropora danai, which is now considered a junior synonym of Acropora abrotanoides, as described in Wallace, 1999 [27], and Veron, 2000 [2]. Acropora nobilis is now considered a junior synonym of A. intermedia, as documented in Wallace, 1999, and Veron et al., 2022 [3,28].

Our study described the status of the reefs in the Jaffna Peninsula, from both a conservation and a bio-geographic point of view. The results of the biogeographic study were relevant because the database of already published work from the Indian Ocean and Red Sea regions indicated that most if not all the species that we recorded were present within these regions [29,30,31,32,33,34,35,36] (

Table 1. Details of scleractinian corals reported to be new to Sri Lanka, with notes on their taxon update details and known geographical distribution (individual closest locations near by Indian territory already clearly presented in our published article).

Sp No.	Family	Genus	Species	Updates on Taxon Details	Geographic Distribution [2,3,28,29,30,31,32,33,34,35,36]
1	Acroporidae Verrill, 1901	Acropora Oken, 1815	Acropora aspera Dana, 1846	3, 28	Red Sea, Indo Pacific, South Pacific Ocean
2			Acropora digitifera Dana, 1846	3, 28	Red Sea, Indian Ocean, Indo Pacific, Mozambique, South Africa, South Pacific Ocean, Madagascar
3			Acropora gemmifera Brook, 1892	3, 28	Red Sea, Indo Pacific, South Pacific Ocean, Mozambique, Federal Republic of Somalia
4			Acropora latistella Brook, 1891	3, 28	Red Sea, Central Indo Pacific, South Africa, Mozambique
5			Acropora pulchra Brook, 1891	3, 28	Red Sea, Indo Pacific, Indian Ocean, Mozambique, South Pacific Ocean
6		Alveopora Blainville, 1830	Alveopora allingi Hoffmeister, 1925	3, 28	Red Sea, Indian Ocean, South Africa, Mozambique, South Pacific Ocean
7		Astreopora Blainville, 1830	Astreopora myriophthalma Lamarck, 1816	3, 28	Red Sea, Indian Ocean, Indo Pacific, Mozambique, South Africa, South Pacific Ocean, Kenya
8			Astreopora listeri Bernard, 1896	3, 28	Red Sea, Indian Ocean, Indo Pacific, Mozambique, South Pacific Ocean, Madagascar, Kenya
9			Astreopora ocellata Bernard, 1896	3, 28	Red Sea, Central Indo Pacific, Indian Ocean
10		Montipora Blainville, 1830	Montipora flabellata Studer, 1901	3, 28	South Pacific Ocean, Mozambique
11			Montipora informis Bernard, 1897	3, 28	Red Sea, Indian Ocean, Indo Pacific, Mozambique, South Pacific Ocean, Madagascar
12	Agariciidae Gray, 1847	Coeloseris Vaughan, 1918	Coeloseris mayeri Vaughan, 1918	3, 28	Red Sea, Central Indo Pacific, South Africa, Mozambique
13	Dendrophylliidae Gray, 1847	Turbinaria Oken, 1815	Turbinaria frondens Dana, 1846	3, 28	Red Sea, Indian Ocean, Indo Pacific, Mozambique, Kenya, New Zealand
14			Turbinaria reniformis Bernard, 1896	3, 28	Red Sea, Indian Ocean, Indo Pacific, Kenya, Federal Republic of Somalia
15			Turbinaria stelluta Lamarck, 1816	3, 28	Indo Pacific, Madagascar, South Pacific Ocean
16	Oulastreidae Vaughan, 1919	Oulastrea Milne Edwards & Haime, 1848	Oulastrea crispata Lamarck, 1816	3, 28	Andaman Nicobar Islands, Western Basin
17	Scleractinia incertae sedis	Pachyseris Milne Edwards & Haime, 1849	Pachyseris gemmae Nemenzo, 1955	3, 28	Andaman Nicobar Islands, Indian Ocean
18	Merulinidae Verrill, 1865	Coelastrea Verrill, 1866	Coelastrea palauensis Yabe & Sugiyama, 1936	3, 28	Red Sea, Central Indo Pacific, Indian Ocean
19		Cyphastrea Milne Wdwards & Haime, 1848	Cyphastrea japonica Yabe & Sugiyama, 1932	3, 28	Philippines, Central Indian Ocean
20			Cyphastrea microphthalma Lamarck, 1816	3, 28	Red Sea, Indian Ocean, Indo West Pacific, Mozambique, Madagascar, South Africa, South Pacific Ocean, Kenya
21		Dipsastraea Blainville, 1830	Dipsastraea amicorum Milne Edwards & Haime, 1850	3, 28	Red Sea, Indian Ocean, Mozambique
22			Dipsastraea rotumana Gardiner, 1899	3, 28	Red Sea, Indian Ocean, Indo West Pacific, Mozambique, South Africa, South Pacific Ocean
23			Dipsastraea lizardensis Veron & Pichon, 1977	3, 28	Red Sea, Central Indo Pacific, Indian Ocean
24		Echinopora Lamarck, 1816	Echinopora gemmacea Lamarck, 1816	3, 28	Red Sea, Indian Ocean, Indo West Pacific, Mozambique, Madagascar, South Africa, South Pacific Ocean, Kenya, Federal Republic of Somalia
25		Goniastrea Milne Edwards & Haime, 1848	Goniastrea minuta Veron, 2000	3, 28	Seychelles, Philippines, South Pacific Ocean
26		Platygyra Ehrenberg, 1834	Platygyra acuta Veron, 2000	3, 28	Indo Pacific, Andaman and Nicobar Islands
27	Lobophylliidae Dai & Horng, 2009	Acanthastrea Milne Edwards & Haime, 1848	Acanthastre ishigakiensis Veron, 1990	3, 28	Andaman and Nicobar Islands, South Pacific Ocean
28		Micromussa Veron, 2000	Micromussa amakusensis Veron, 1990	3, 28	Western Indian Ocean, Federal Republic of Somalia
29	Poritidae Gray, 1842	Porites Link, 1807	Porites evermanni Vaughan, 1907	3, 28	Andaman and Nicobar Islands, Indo Pacific
30			Porites murrayensis Vaughan, 1918	3, 28	Red Sea, Indian Ocean, Indo Pacific, South Africa
31			Porites pukoensis Vaughan, 1907	3, 28	Indian Ocean, Madagascar
32		Goniopora de Blainville, 1830	Goniopora lobata Milne Edwards, 1860	3, 28	Red Sea, Indian Ocean, Indo Pacific, Mozambique, South Pacific Ocean, Federal Republic of Somalia
33			Goniopora minor Crossl &, 1952	3, 28	Red Sea, Central Indo Pacific, Indian Ocean
34			Goniopora somaliensis Vaughan, 1907	3, 28	Red Sea, Indian Ocean, Indo Pacific, Mozambique, South Africa, South Pacific Ocean, Kenya, Federal Republic of Somalia
35			Goniopora tenuidens Quelch, 1886	3, 28	Indian Ocean. Indo Pacific, South Pacific Ocean
36	Siderastreidae Vaughan & Wells, 1943	Siderastrea Blainville, 1830	Siderastrea savignyana Milne Edwards & Haime, 1850	3, 28	Red Sea, Indian Ocean, Indo Pacific, Mozambique, Madagascar, South Pacific Ocean

). While acknowledging the fact that Hendawitharana et al. (2023) found our tile misleading, we would like to state the following. The phrase “Confirmed by DNA-Bar coding” referred only to the genetic confirmation of the eight newly recorded species studied in this way to check their identity. We do not believe that the title misleads the readership and trust that disagreement on this point can be disregarded. We accept the possibility that a few of the identifications based on confirmation by barcoding may turn out not to be as robust as we would hope, but we do stand by the validity of the identifications described in our paper.

In summary, our study holds significant value in its exploration of previously unstudied taxa and geographic regions, as well as in shedding light on the potential existence of new species records, albeit with acknowledged methodological limitations. Moreover, through this initial investigation, our goal was to raise awareness regarding the area’s potential significance and to propose further research with enhanced methodologies for robustly validating our discoveries. We also recognize the challenges associated with conducting comprehensive species occurrence inventories and the circumstances where their approaches may prove beneficial.