diversity Review Cryptic Clitellata: Molecular Species Delimitation of Clitellate Worms (Annelida): An Overview Svante Martinsson * and Christer Erséus Systematics and Biodiversity, Department of Biological and Environmental Sciences, University of Gothenburg, Box 463, SE-405 30 Göteborg, Sweden; [email protected] * Correspondence: [email protected] Abstract: Methods for species delimitation using molecular data have developed greatly and have become a staple in systematic studies of clitellate worms. Here we give a historical overview of the data and methods used to delimit clitellates from the mid-1970s to today. We also discuss the taxonomical treatment of the cryptic species, including the recommendation that cryptic species, as far as possible, should be described and named. Finally, we discuss the prospects and further development of the field. Keywords: DNA-barcoding; Oligochaeta; cryptic species 1. Introduction Species delimitation, i.e., the process of determining species boundaries and discover- ing species, is a field that has developed quickly since the introduction of genetic data [1,2]. The development has been both on the data side, from protein patterns to large genomic datasets, and on the analytical side, from clustering and measures of genetic distances to complex analyses based on coalescent theory. These advances have led to an increase in Citation: Martinsson, S.; Erséus, C. the discovery of cryptic species, i.e., species that are morphologically similar and, therefore, Cryptic Clitellata: Molecular Species have been classified as the same nominal species [3]. Cryptic species are found all over Delimitation of Clitellate Worms the animal kingdom (e.g., [4,5]), including annelids (e.g., [6,7]) and, despite morphological (Annelida): An Overview. Diversity similarities, they may differ in ecologically and physiologically important aspects (see, 2021, 13, 36. https://doi.org/ e.g., [8,9]). Species are basic biological units and entities of generalisation, and, therefore, 10.3390/d13020036 the basis of most studies. A number of clitellate species are used as models in several fields, e.g., ecotoxicology, neurobiology and soil ecology [10,11] and, in several of the species used, Received: 17 December 2020 taxonomical problems have been found [12–16]. In this kind of work, it is important to Accepted: 18 January 2021 Published: 20 January 2021 know the true identity of the organisms to be able to compare the results between studies, and to correctly generalise the findings to species level, and to understand the functional differences between the taxa in question. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Clitellata is a large “class” of segmented worms, comprising about one third of all published maps and institutional affil- known annelid species. It is placed within “subclass” Sedentaria (e.g., [17,18]), which is iations. often thought of as a (major) polychaete group. Clitellates seem to have evolved in the transitional zones between marine and continental waters [19], and a majority of the species live within soil or aquatic sediment [20]. Unlike polychaetous annelids, they lack parapodia, and their prostomium lacks appendages. The monophyly of Clitellata is strongly supported by their unique mode of reproduction. Clitellates are hermaphrodites and characterized Copyright: © 2021 by the authors. by the “clitellum”, an epidermal structure, secreting a protective cocoon for the embryos, Licensee MDPI, Basel, Switzerland. This article is an open access article which develop without a larval stage (see, e.g., [21]). The external morphology of clitellates distributed under the terms and is rather stable and offers few characters trustworthy of the taxonomic separation of conditions of the Creative Commons taxa. The shape, position and number of gonads have historically been of fundamental Attribution (CC BY) license (https:// importance for the classification [22]. The burrowing and interstitial habitats of most creativecommons.org/licenses/by/ clitellates are likely to be the reason for their conserved morphology, as the evolutionary 4.0/). pressures in these environments may favour morphological stasis [3,9,23]. Due to the Diversity 2021, 13, 36. https://doi.org/10.3390/d13020036 https://www.mdpi.com/journal/diversity Diversity 2021, 13, 36 2 of 13 lack of externally discernible characters, many clitellates are hard to delimit and identify without the aid of molecular markers, and their species diversity has, in many cases, been underestimated when based on morphology alone (many examples will be given below). This fact has led to the rise of molecular approaches to separate species, which we will explore in this review. Species delimitation can be divided into two steps, species discovery and species vali- dation [24]. In the first step, the researchers form hypotheses about the species boundaries, which are then tested in the second step. In the species discovery phase, typically a single data source, e.g., morphology or DNA-barcoding, is used. Testing these hypotheses in the species validation step are often based on additional data and more sophisticated analyses. In most studies, this division between species discovery and validation is not explicitly stated, but rather implied. The definition of cryptic species varies between researchers. Some use a relaxed definition. They count all cases as cryptic where species fall within the morphological variation of the same nominal species, even when there are minor differences between them, e.g., [3]. Others use a stricter definition and distinguish between true cryptic and pseudo-cryptic species, where the first refer to species between which no morphological differences are observed, while the latter are species that do show some differences, but still are so similar that they would be classified as the same nominal species based on morphology (e.g., [25]). In this paper, we apply the broader definition of cryptic species. Moreover, we use a liberal definition of molecular species delimitation. We include papers that explore molecular data to support species also discriminated morphologically, even if the authors do not explicitly test species limits. In this paper, we aim to give an overview of the research field of species delimitation, and cryptic species, in Clitellata. We will examine the development of methods and the new data used in delimitation of clitellate species and discuss some of the problems arising when describing cryptic species. Finally, we will consider possible directions for this field. 2. History of the Field Here we present an historical overview over the field of molecular species delimitation of clitellate worms, from a first publication in the 1970s to papers published in 2020. In total, 104 studies where found (Figure1, Table S1). We identified four categories of data studied and have structured the overview accordingly, dividing this section into methods categorized as: (1) gel electrophoresis of proteins; (2) non-sequenced DNA; (3) Sanger- sequencing of a limited number of DNA fragments; (4) High-Throughput Sequencing (HTS) of a large number of DNA fragments. This classification is somewhat arbitrary, and Diversity 2021, 13, x FOR PEER REVIEW 3 of 14 methods from more than one category have been used together in many instances and is schematically shown in Figure1. Figure 1. Historical timeline of the development of molecular species delimitation in Clitellata, showing the year of the first Figure 1. Historical timeline of the development of molecular species delimitation in Clitellata, showing the year of the firststudy, study, and and the totalthe total number number of studies, of studies, of the of the four four major major categories categories of methods of method referenceds referenced in thisin this paper paper (see (see Table Table S1 forS1 fordetails). details). The The histogram histogram shows shows the the total total number number of studiesof studies (all (all categories) categories) per per year. year. 2.1. Protein Gel Electrophoresis The first publications on the species delimitation of clitellates, by means of molecular data, explored variation in proteins revealed by gel electrophoresis. In these molecular methods, proteins encoded by alleles at some locus (alloenzymes), or proteins with the same function but encoded by separate genes at different loci (isoenzymes) are separated on gels, and the pattern observed is used to infer the separation of populations. The first works by using protein gel electrophoresis to explicitly test species hypotheses of clitel- lates that occurred in the 1970s and 1980s, (e.g., [26–28]), although the implication of this method was discussed already by Milbrink and Nyman [29], who saw it mainly as a sup- plement to morphological identification of species in ecological studies. Isoenzymes and alloenzymes continued to be used, often in combination with other methods (e.g., [30– 35]). Another gel-based method is the study of general protein patterns, where a mix of proteins extracted from a specimen is run on a gel, producing a banding pattern that is then compared between individuals. The pattern produced is assumed to be species spe- cific and an index based on protein patterns was suggested [36], which was then mainly used for studies of the family Enchytraeidae [30,33,35,37,38]. Crossed immunoelectropho- resis (CIE) is another method that, to our knowledge, was only tested once in clitellate systematics—i.e., to separate populations