diversity Article DNA Barcoding of Marine Mollusks Associated with Corallina officinalis Turfs in Southern Istria (Adriatic Sea) Moira Burši´c 1, Ljiljana Iveša 2 , Andrej Jaklin 2, Milvana Arko Pijevac 3, Mladen Kuˇcini´c 4, Mauro Štifani´c 1, Lucija Neal 5 and Branka Bruvo Madari´c¯ 6,* 1 Faculty of Natural Sciences, Juraj Dobrila University of Pula, Zagrebaˇcka30, 52100 Pula, Croatia; [email protected] (M.B.); [email protected] (M.Š.) 2 Center for Marine Research, Ruder¯ Boškovi´cInstitute, G. Paliage 5, 52210 Rovinj, Croatia; [email protected] (L.I.); [email protected] (A.J.) 3 Natural History Museum Rijeka, Lorenzov Prolaz 1, 51000 Rijeka, Croatia; [email protected] 4 Department of Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000 Zagreb, Croatia; [email protected] 5 Kaplan International College, Moulsecoomb Campus, University of Brighton, Watts Building, Lewes Rd., Brighton BN2 4GJ, UK; [email protected] 6 Molecular Biology Division, Ruder¯ Boškovi´cInstitute, Bijeniˇcka54, 10000 Zagreb, Croatia * Correspondence: [email protected] Abstract: Presence of mollusk assemblages was studied within red coralligenous algae Corallina officinalis L. along the southern Istrian coast. C. officinalis turfs can be considered a biodiversity reservoir, as they shelter numerous invertebrate species. The aim of this study was to identify mollusk species within these settlements using DNA barcoding as a method for detailed identification of mollusks. Nine locations and 18 localities with algal coverage range above 90% were chosen at four research areas. From 54 collected samples of C. officinalis turfs, a total of 46 mollusk species were Citation: Burši´c,M.; Iveša, L.; Jaklin, identified. Molecular methods helped identify 16 gastropod, 14 bivalve and one polyplacophoran A.; Arko Pijevac, M.; Kuˇcini´c,M.; Štifani´c,M.; Neal, L.; Madari´c,B.B.¯ species. COI sequences for two bivalve species (Musculus cf. costulatus (Risso, 1826) and Gregariella DNA Barcoding of Marine Mollusks semigranata (Reeve, 1858)) and seven gastropod species (Megastomia winfriedi Peñas & Rolán, 1999, Associated with Corallina officinalis Eatonina sp. Thiele, 1912, Eatonina cossurae (Calcara, 1841), Crisilla cf. maculata (Monterosato, 1869), Turfs in Southern Istria (Adriatic Sea). Alvania cf. carinata (da Costa, 1778), Vitreolina antiflexa (Monterosato, 1884) and Odostomia plicata Diversity 2021, 13, 196. https://doi. (Montagu, 1803)) represent new BINs in BOLD database. This study contributes to new findings org/10.3390/d13050196 related to the high biodiversity of mollusks associated with widespread C. officinalis settlements along the southern coastal area of Istria. Academic Editor: Gert Wörheide Keywords: mollusks; gastropods; bivalves; Corallina officinalis; molecular identification; DNA bar- Received: 1 March 2021 coding; Adriatic Sea Accepted: 29 April 2021 Published: 30 April 2021 Publisher’s Note: MDPI stays neutral 1. Introduction with regard to jurisdictional claims in published maps and institutional affil- Corallina officinalis L. is an encrusting red alga, globally widely distributed in the iations. temperate zone where it forms dense settlements in the intertidal area. It modifies the environment through its characteristic structure and is an ideal example of how algae affect the composition and interaction of organisms within their settlements [1]. It shelters many macrofaunal groups from wave action, predation, and stress from desiccation in the intertidal area [2–4]. Based on the mapping results of its habitats along the eastern Adriatic Copyright: © 2021 by the authors. Corallina Licensee MDPI, Basel, Switzerland. coast [5], the coastal area of the Adriatic Sea is a suitable location for ’s settlements. This article is an open access article Looking on a smaller scale, along the western Istrian coast, the same continuous widespread distributed under the terms and distribution is recorded for Corallina officinalis and Ellisolandia elongata (J.Ellis & Solander) conditions of the Creative Commons K.R.Hind & G.W.Saunders, with C. officinalis dominating in areas with no anthropogenic Attribution (CC BY) license (https:// impact [6]. creativecommons.org/licenses/by/ Changes in the structure and composition of macrofauna found in C. officinalis turfs 4.0/). have been studied worldwide [2,7–11]. All previous data showed high abundance and Diversity 2021, 13, 196. https://doi.org/10.3390/d13050196 https://www.mdpi.com/journal/diversity Diversity 2021, 13, x FOR PEER REVIEW 2 of 17 Changes in the structure and composition of macrofauna found in C. officinalis turfs Diversity 2021, 13, 196 have been studied worldwide [2,7–11]. All previous data showed high abundance2 ofand 17 variety of invertebrates present. The most common taxonomic groups found were polychaetes, crustaceans (amphipods, isopods, tanaidaceans, decapods), pycnogonids varietyand mollusks, of invertebrates mostly represented present. The by mostbivalves common and gastropods taxonomic [2,8,12–24]. groups found Notably, were poly-most chaetes,of the invertebrate crustaceans specimens (amphipods, recorded isopods, in tanaidaceans,C. officinalis turfs decapods), were present pycnogonids as juvenile and mollusks,stages, making mostly the represented identification by bivalves according and to gastropods morphological [2,8,12 features–24]. Notably, very complex most of the[2,8,12–24]. invertebrate specimens recorded in C. officinalis turfs were present as juvenile stages, makingDNA the barcoding identification [25] accordingcan be very to morphologicaluseful in identifying features specific very complex taxonomic [2, 8groups,12–24]. or developmentalDNA barcoding stages [ 25of] various can be veryinvertebrate useful ins in identifying situations specificwhere species taxonomic determination groups or developmentalbased on morphology stages of is various extremely invertebrates problematic in situations [26,27], as where well species as in determinationcases where a baseddamaged on morphology organism is is present extremely or just problematic a tissue [fragment26,27], as is well available as in cases [28]. where DNA a barcoding damaged organismof marine is organisms present or has just been a tissue ongoing fragment arou isnd available the world [28]. for DNA more barcoding than a ofdecade marine to organismsdistinguish has cryptic been ongoingand invasive around species the world as well for as more to help than aexplain decade different to distinguish puzzling cryptic life andcycles, invasive making species it a convenient as well as tool to helpfor assessm explainent different and conservation puzzling life of marine cycles, biodiversity making it a convenient[29–32]. However, tool for assessmentit has not yet and been conservation used in ofstudies marine of biodiversity invertebrate [29 fauna–32]. However,within C. itofficinalis has not turfs, yet been and used this paper in studies presents of invertebrate how sequence fauna data within of theC. mitochondrial officinalis turfs, cox-1 and gene this paper(i.e., the presents barcode how region, sequence mtDNA data ofCOI-5P) the mitochondrial can be usedcox-1 to confirmgene (i.e., or theeventually barcode region,correct mtDNAmorphological COI-5P) identifications can be used to for confirm such problematic or eventually specimens. correct morphological Additionally, identifications this study is forone suchof the problematic first examples specimens. of barcode Additionally, methodology this usage study for is Croatian one of the marine first examplesmollusks. ofWe barcode further methodology highlight the usage importance for Croatian of molecular marine mollusks. methods Wein furthermarine highlightinvertebrates the importanceidentification of molecularand supplementing methods in marineexisting invertebrates DNA barcode identification databases and such supplementing as BOLD existingdatabase DNA with barcodevalid sequence databases data. such as BOLD database with valid sequence data. 2. Materials and Methods 2. Materials and Methods The research was carried out in the northeastern part of the Adriatic Sea, in the coastal The research was carried out in the northeastern part of the Adriatic Sea, in the area of southern Istria and Brijuni National Park. Intertidal area of southern Istria is coastal area of southern Istria and Brijuni National Park. Intertidal area of southern Istria dominated by carbonate sedimentary rocks which represents a desirable substrate type is dominated by carbonate sedimentary rocks which represents a desirable substrate type for the settlements of the red alga Corallina officinalis [33]. Sampling was performed in the for the settlements of the red alga Corallina officinalis [33]. Sampling was performed in the summer period of 2018, between June and August, in four different sampling areas named summer period of 2018, between June and August, in four different sampling areas named Pula, Banjole, Premantura and NP Brijuni (Brijuni National Park). Each sampling area Pula, Banjole, Premantura and NP Brijuni (Brijuni National Park). Each sampling area had had two or, in case of NP Brijuni, three sampling localities with a total of nine sampling locationstwo or, in indicated case of inNP Figure Brijuni,1. three sampling localities with a total of nine sampling locations indicated in Figure 1. FigureFigure 1.1. Sampling locations (in (in italics) italics) within within four four ar areaseas (Pula, (Pula, Banjole, Banjole, Pr Premantura,emantura, NP NP Brijuni) Brijuni) situatedsituated