﻿Taxonomic revision of the Nitocraaffinis Gurney, 1927 species complex (Harpacticoida, Ameiridae) with descriptions of four new species and re-evaluation of its subspecies

﻿Abstract Due to the recent increasing importance of microcharacters in copepod taxonomy, it has become evident that many species lacking detailed descriptions actually constitute to a species complex. In this study, Nitocraaffinis is redescribed based on lectotype material from Lake Timsah (Egypt) which facilitated a thorough detailed comparison with specimens of N.affinis recorded from distantly related localities. The results unequivocally revealed that the specimens of N.affinis examined in this study belong to a different species. As a result, four new species, Nitocrasonmezisp. nov. and Nitocraserdarsakisp. nov. from the Turkish coast, Nitocraalperisp. nov. from the Indian Ocean, and Nitocraloweaesp. nov. from Brighton, England are herein described as new to science. On the other hand, all subspecies of N.affinis, namely N.affinisrijekana Petkovski, 1954, N.affiniscalifornica Lang, 1965, N.affinisstygia, Por. 1968, and N.affiniscolombiensis Fuentes-Reinés & Suárez-Morales, 2014 are elevated to species rank. An updated key the species of the affinis group is also provided.


Introduction
The family Ameiridae Boeck, 1865 is ranked third in terms of number of taxa in the Harpacticoida, after Miraciidae Dana, 1846 and Canthocamptidae Sars, 1906, comprising 47 genera and up to 300 species (Corgosinho et al. 2020).Boeck (1865) originally established both Ameira Boeck, 1865 and Nitocra Boeck, 1865 with limited descriptions.The genus Nitocra is represented by 81 valid species and subspecies (Karanovic et al. 2015;Huys 2021;Fuentes-Reinés et al. 2022).The taxonomy of the genus Nitocra, similar to numerous other genera within the family, has posed challenges due to the lack of detailed species descriptions and taxonomic information (Karanovic and Pesce 2002).Initially designated as "Nitokra" by Boeck (1865), the nomenclature was later amended to "Nitocra" (Giesbrecht 1881), a spelling upheld until Bowman (1988) highlighted its erroneous usage.This viewpoint was endorsed by Wells (2007), referencing ICZN (1999: § 33.3.1),which advocates for prevailing name usage, thereby asserting the adoption of "Nitocra".
Nitocra affinis was originally described by Gurney (1927) from Ismailia and Port Tawfiq (Egypt), collected during the Cambridge Expedition to the Suez Canal (1924), who described both sexes based on an undisclosed number of specimens.The distributional range of N. affinis has expanded with the description of several subspecies from different ecological environments: N. affinis rijekana Petkovski, 1954 was described from the North Adriatic, N. affinis stygia Por, 1968 was described from the Red Sea, N. affinis californica Lang, 1965 was described from California, and N. affinis colombiensis Fuentes-Reinés & Suárez-Morales, 2014 was described from a coastal lagoon in Colombia (Petkovski 1954;Lang 1965;Por 1968;Fuentes-Reinés and Suárez-Morales 2014).
Considering the distributional range of the species, including its subspecies, the wide morphological variations observed among the populations, and the diversity in their ecological habitats, postulations arise that more than one species (i.e., a species complex) may exist under the name of N. affinis, and hence needing an urgent revision.Here, N. affinis is partially redescribed based on the only incomplete female specimen collected and deposited at the collection of the NHMUK by R. Gurney himself, and proposed here as lectotype (see below), and several populations previously identified as N. affinis from a wide range of habitats and geographic localities were re-examined in detail to morphologically delineate the specific range.

Nitocra affinis Gurney, 1927 Figs 1, 2
Unverified records.Bermuda (Willey 1930), Italia (Chappuis 1938), Sweden (Lang 1935), England (Gurney 1932), Federated States of Micronesia (Vervoort 1964).Type material.Lectotype: Egypt • 1 ♀; Ismailia.Mounted on one slide.Damaged.Abdomen lost.Gurney, R leg.;NHMUK reg. no. 1928.4.2.107. Gurney (1927) recorded N. affinis from both Ismailia and Port Tawfiq and described both sexes based on an undisclosed number of specimens.Since a holotype was not designated by Gurney (1927) all specimens collected from both localities are collectively regarded as the type series.The incomplete female specimen collected and identified by R. Gurney (incorrectly labelled as a cotype) and deposited in the NHMUK under reg. no. 1928.4.2.107 is the only surviving syntype and is here designated as the lectotype of N. affinis.The place of origin of the latter is Lake Timsah, Ismailia which becomes the type locality of N. affinis according to ICZN Art.76.2.
Antennule (Fig. 1C) eight-segmented and joined to the cephalothorax with small triangular pseudosegment (arrowed in Fig. 1C); first segment with spinules at the ventrolateral margin and with a tube pore near the inner dorsal margin, and with row of slender spinules on ventral surface.Long, slender aesthetasc on fourth segment fused basally with adjacent large seta.Two lateral setae on seventh segment and four lateral setae on eighth segment biarticulate at base.Setal formula 1- Maximum length/maximum width ratio of antennular segments as 1:1.2:1.3:1.8:1.2:1.8:1.3:2.8.
Rostrum (Fig. 1D).Small with two dorsal sensilla near the base of apical rostral projection, which is ~ half of the rostral length, with an opening (pore) distally.
Antenna (Fig. 1E).Coxa small, unornamented.Basis and proximal endopodal segment fused forming allobasis (ancestral segmentation between basis and first endopodal segment visible near base of exopod) with a spinule row near the base of exopod.Exopod one-segmented, with two unipinnate spines and one slender naked seta; endopod lost.
Mandible (Fig. 1F).Gnathobase with coarse teeth ventrally, dorsal unipinnate seta could not be observed due to natural position of structure.Uniramous palp two-segmented comprising basis and one-segmented endopod.Basis armed with a bipinnate spine.Endopod with one plumose lateral seta, and four naked setae (two of them basally fused).
Maxillule not observed.Note: this appendage was impossible to be reliably observed in detail because its position underneath the maxilla.But the structure and setation of the maxillule agrees with the that of N. loweae sp.nov.On the other hand, the lectotype material was too fragile and the mouth parts were too small; therefore, the only specimen was not dissected.The maxillule had better be redescribed based on newly collected materials, preferably from newly collected topotype.
Maxilla (Fig. 1G) with two well-developed endites on the syncoxa with a robust row of spinules on outer margin; distal endite with a strong unipinnate seta and two naked setae; proximal endite small, with two naked setae.Allobasis transformed into claw, with one naked seta at base.Endopod a reduced segment with one seta.The maxilla should be redescribed based on newly collected topotypes.
Maxilliped (Fig. 1H) subchelate.Syncoxa unornamented with one subapical plumose seta.Basis ~ 2.4 × as long as maximum width, with row of spinules along inner margin and three small spinules on outer distal corner.Endopod represented by strong claw accompanied at base by a minute naked seta.
P1 (Fig. 2A).Coxa rectangular, outer distal margin ornamented with fine setules; anterior surface with a row of spinules.Basis with spinule row at the base of strong pinnate inner spine; with spinule row along distal margin.Inner margin of basis with robust spinules.Exp-1,2 with outer pinnate spine.Exp-2 with an inner plumose seta.Exp-3 with two geniculate apical setae and three outer pinnate spines.Enp-1 longer than exopod, 5 × as long as maximum width and ornamented with row of fine spinules on the middle of the segment along inner  margin, with three spinules on distal margin and with a bipinnate spine originating from the distal half of the segment; enp-2 with a plumose seta on inner corner and with spinules on outer margin; enp-3 with one plumose seta and two geniculate setae distally and with few spinules on outer margin.
Rostrum (Fig. 5A) small, triangular, without clear demarcation between the distal and the proximal part of rostrum (cf.Fig. 1C of N. affinis) with tube pore distally and with two dorsal sensilla (Fig. 5A).
Antenna (Fig. 5B) comprising coxa, allobasis, one-segmented endopod and one-segmented exopod.. Coxa very short and unornamented.Allobasis cylindrical, ~ 2.7 × as long as maximum width, ornamented proximally with three spinule rows.Free endopodal segment with proximal part narrower than distal part, ~ 2.5 × as long as its maximum width, ornamented with surface frill subdistally, and with longitudinal row of spinules along inner margin, with another spinule row near the base of two lateral unipinnate spines flanking thin naked seta; apical armature consisting of five geniculate setae, one of them fused basally to additional unipinnate non-geniculate seta.Exopod with narrow proximal half and somewhat wider distal part, ~ 2.5 × as long as its maximum width, unornamented, armed with two curved, strong unipinnate apical setae and one spinulose subdistal seta, the latter longest.
Maxillule (Fig. 5D).Praecoxa large with few spinules.Praecoxal arthrite rectangular; with two setae on anterior surface, lateral spinule row and distal armature consisting of four spines (two of which with apical combs) and one unipinnate seta.Coxal endite shorter than praecoxal arthrite, with long distally curved spine and three slender naked setae.Basis rectangular, with five slender naked setae on distal margin.Endopod represented by minute but distinct segment, unornamented and armed with two plumose apical setae.Exopod absent.
Maxilla (Fig. 5E).Syncoxa with spinule row and two well-developed (coxal) endites; proximal endite somewhat bulbous, expanded distally and armed with two plumose setae; distal endite cylindrical with two naked apical setae of equal in length.Allobasis transformed into claw-like pinnate endite; with a pinnate seta at base and with few spinules along convex margin near the base of endopod.Endopod represented by two slender naked setae equal in length.Maxilliped (Fig. 5F) subchelate.Syncoxa with one plumose subapical seta and with spinule rows on posterior surface.Basis ~ 2.6 × as long as maximum width with row of spinules along inner margin and with row of spinules on outer distal corner.Endopod represented by strong claw accompanied at base by minute naked seta.
P1 (Fig. 6A).Coxa with anterior row of spinules as figured and few spinules along inner margin.Basis with fine setules along inner margin, and with spinule row near the base of short pinnate inner spine located at the base of endopod.Outer basal bipinnate spine located close to the exopod, overlaid by a row of strong spinules.Exp-1 smallest segment, carrying row of strong spinules on outer margin and subapically one bipinnate outer spine.Exp-2 also with an outer row of strong spinules, a bipinnate outer spine, and a long plumose seta on inner margin.Exp-3 with fine setules near inner distal margin, with one geniculate semi-plumose apical seta, one geniculate naked distal seta and three pinnate outer spines.Enp-1 longer than exopod, ~ 3 × as long as maximum width, and ~ 2 × as long as enp-1, 2 combined; inner margin with long longitudinal spinules; distal margin with four robust spinules, and a long plumose seta on inner subdistal margin; enp-2, 3 squarish and equal in length; enp-2 with a long, plumose seta on inner margin, and with two spinules on outer distal margin; enp-3 with one plumose inner seta, one apical unipinnate distal geniculate seta, and one apical unipinnate outer seta, outer margin with a row of spinules.
P2-P4 (Fig. 6C, D, F).Coxa with row of posterior spinules near outer margin and with an anterior pore near inner distal corner.Basis triangular, ornamented with spinule row along distal margin between the base of exopod and endopod, with anterior spinules on outer corner near the base of outer seta/spine.Exp-1, 2 with anterior spinules near the base of outer spine extending to the outer margin of the segment.Exp-1, 2 and enp-1, 2 with hyaline frills along inner distal margin.All endopodal segments covered with robust spinules along outer margin.Exp-2, 3 and enp-2 with a pore on anterior surface.P2-P4 exp-2 with one plumose seta; P2, P3 exp-3 with seven, P4 exp-3 with eight elements.P5 (Fig. 4D).Baseoendopod with five spinulose setae (slightly fringed at tip) along distal margin and with setules along inner margin, with two anterior pores (one near the base of outer basal seta and the other one near the distal margin; outer basal seta plumose.Exopod with one anterior pore distally, with setules along inner margin and with robust spinules along distal margin, with six setae (setae I-VI, numbered from inner to outer margin respectively), setae I, IV, and V pinnate; setae II, III, and VI naked; seta II is the longest; outer margin of exopodal lobe with one tube pore, and with double spinules group.
Inner distal seta of P3 exp-3 (Fig. 6E) slender and shorter than in female.P5 (Fig. 4C) baseoendopod armed with five spinulose setae (four of them equal in length, the second inner seta longest and 1.5 × as long as the other setae) and with two pores on anterior surface; exopod with six setae (outer margin with one naked seta (seta VI) and one spinulose seta (seta V), apical margin with one naked (seta IV) and two spinulose setae (setae II and III), inner margin with one long plumose seta (seta I) (abnormal seta of one leg arrowed in Fig. 4C; the same seta of the other leg normal), with four strong spinules along outer proximal margin and with two or three spinules along inner proximal margin.
P6 (Fig. 4A) asymmetrical, only one leg functional; each leg with two naked outer setae and short inner plumose robust seta.
Etymology.The specific name is given in honour of Dr Miranda Lowe for her contribution to copepod taxonomy as a curator of the Crustacea collection of The Natural History Museum of London.It is a noun in the genitive case.
Description (adult female holotype).Body (Fig. 9A) semicylindrical; total body length measured from tip of the rostrum to posterior end of the caudal rami 400 μm (n = 1).Surface sensilla and pores as figured (Fig. 9A, B).Urosomites with finely serrated hyaline frills, and with complex spinule rows as figured (Fig. 9A-C).Genital double-somite (Fig. 9A, C) viewed as squarish in dorsal and ventral view, rectangular in lateral view (Fig. 9B), with lateral suture line; with two continuous spinule rows dorsally extending laterally as figured.Anal somite (Fig. 9A, C) with two pores located ventrolaterally and medially, anal operculum with fifteen robust spinules.Caudal rami (Fig. 9A, C) short and squarish; with fine setules near the base of seta VII, with row of spinules dorsally near the base of seta II; few spinules present around inner distal margin; with seven setae: seta I minute; seta II slightly displaced dorsally; setae IV and V well-developed and pinnate; seta VI located near inner distal margin and naked; seta VII tri-articulate at base.Antenna (Fig. 10A) comprising coxa, allobasis, one-segmented endopod and one-segmented exopod same as in N. loweae sp.nov.except for allobasis without spinule rows proximally.Endopod without longitudinal row of spinules along inner margin.Exopod with a weakly pinnate subdistal seta.
Maxilla (Fig. 10C) with syncoxa and two well-developed endites same as in N. loweae sp.nov.except for syncoxa without spinule row; distal endite of syncoxa cylindrical with one naked, and one stronger and longer semi-pinnate apical setae, allobasis without spinules along convex margin near the base of endopod.
P1 (Fig. 11A) same as in N. loweae sp.nov.except for basis without spinules along inner margin; inner margin of exp-1, 2 unornamented; innermost geniculate seta of exp-3 naked; exopod reaching slightly above the middle of enp-1 and aligned with the insertion of the inner seta of enp-1; enp-1 ~ 4.3 × as long as maximum width; subdistal seta of enp-1 unipinnate and located more proximally than that of N. loweae sp.nov.enp-2 without spinules on outer distal margin; enp-3 with two small spinules on outer margin.P2 (Fig. 11B) similar to that of N. loweae sp.nov.except for exp-2 without setules along inner margin; inner seta of enp-3 unipinnate and stronger than in N. loweae sp.nov.
P3 (Fig. 11C) similar to that of N. loweae sp.nov.except for exp-2 with setules along inner margin; innermost seta of enp-3 unipinnate and stronger than in N. loweae sp.nov.P4 (Fig. 11D) similar to that of N. loweae sp.nov.except for exp-2 without setules along inner margin; middle inner seta of exp-3 unipinnate, longest and stronger than that of N. loweae sp.nov.; innermost seta of enp-3 longest and plumose.
P5 (Fig. 9D) similar to that of N. loweae sp.nov.except for inner baseoendopod lobe narrower and extends halfway along the exopod, innermost seta shortest (seta I); outermost seta of exopod (seta VI) longer than in N. loweae sp.nov.
Description (adult female holotype).Body (Fig. 12A, B) similar to N. loweae sp.nov., except for total body length 476 μm (n = 1) measured tip of the rostrum to posterior end of the caudal rami.Pores and sensilla as figured (Fig. 12A, B).
Anal somite (Figs 12A, 13A, B), with anal operculum bearing seventeen posterior spinules; with row of robust spinules flanking each side of the anal operculum; with a posterior row of small spinules, and two pair of pores on ventrally.
Antenna (Fig. 13C) similar to that of N. loweae sp.nov.except for allobasis with spinules only on midway inner margin; subdistal seta of exopod weakly pinnate.
Mandible (Fig. 13D) similar to that of N. loweae sp.nov.except for exopod with four naked apical setae (two of them basally fused at base) and without spinules.
Maxilla (Fig. 13E) similar to that of N. loweae sp.nov.except for allobasis without spinules along convex margin near the base of endopod; endopod with one long naked seta.
P1 (Fig. 14A) similar to that of N. loweae sp.nov.except for coxa without spinules on/near inner margin; basis without setules along inner margin; exopod slightly extends the enp-1; enp-1 ~ 2.6 × as long as maximum width, inner margin with less spinules along inner margin, subdistal seta unipinnate and located more proximally than in N. loweae sp.nov.; inner margin of enp-2 with one setule, outer distal margin with few fine spinules not extending to inner margin.P2 (Fig. 14B) similar to that of N. loweae sp.nov.except for intercoxal sclerite with spinules on anterior surface; basis with setules along inner margin; inner seta of enp-3 uni-plumose and stronger than in N. loweae sp.nov.P3 (Fig. 14C) similar to that of N. loweae sp.nov.except for intercoxal sclerite with spinules on anterior surface; basis with setules along inner margin; exp-2 with setules along inner margin; innermost seta of enp-3 unipinnate and stronger than in N. loweae sp.nov.P4 (Fig. 14D) similar to that of N. loweae sp.nov.except for exp-1 with two fine setules along inner margin; middle inner seta of exp-3 bipinnate distal half, longest and stronger than that of N. loweae sp.nov.; innermost seta of enp-3 uni-plumose and stronger than in N. loweae sp.nov.P5 (Fig. 14E) similar to that of N. loweae sp.nov.except for inner baseoendopod lobe reaching middle of the exopod; exopod tapering apically, and ~ 1.5 × as long as maximum width, outermost seta much longer, seta next to outermost seta slender and naked.Description (adult male holotype): Body (Fig. 15A, B) semi-cylindrical, total body length measured from tip of the rostrum to posterior end of the caudal rami 582 μm (n = 1).Sensilla and pores as figured (Fig. 15A-D).Rostrum small, with two sensilla on distal margin, without rostral extension apically (Fig. 15E).Anal somite with two sensilla on both sides of anal operculum; posterior end covered with robust spinules; inner distal and lateral margin with small spinules and a pair of pores medially on ventral surface.Anal operculum (Fig. 15C) with eleven robust spinules along posterior margin.Caudal rami small and squarish; with transverse fine setules dorsally extending inner margin dorsally, and with a row of spinules laterally; ventrally with pores on near anterior and posterior margin (Fig. 15D).
Antennule (Fig. 16A).Setal pattern and structure similar to that of N. loweae sp.nov.except for segments weaker developed than in N. loweae sp.nov.
Antenna (Fig. 16B) comprising coxa, allobasis, one-segmented endopod and one-segmented exopod similar to that of N. loweae sp.nov.except for allobasis with spinules only on the middle of inner margin; spinule row along inner margin of free endopodal segment; more sparsely distributed than in N. loweae sp.nov.; inner apical seta of free endopodal segment 1.5 × as long as the adjacent apical seta; subdistal seta of exopod weakly pinnate.
Mandible (Fig. 16C) similar to that of N. loweae sp.nov.except for exopod with four naked apical setae (two of them fused basally) and without spinules.
Maxilla (Fig. 16D) similar to that of N. loweae sp.nov.except for allobasis without spinules along convex margin near the base of endopod; endopod with one long seta.
Maxilliped (Fig. 16E) similar to that of N. loweae sp.nov.except for syncoxa ~ 2.6 × as long as maximum width; basis ~ 2.7 × as long as maximum width.
Maxillule similar to that of N. loweae sp.nov.P1 (Fig. 17A) similar to that of N. loweae sp.nov.except for exopod extends the level of inner seta of enp-1; enp-1 ~ 4.5 × as long as maximum width, inner margin with four well-developed spinules along inner margin, subdistal unipinnate seta located more proximally than in N. loweae sp.nov.; exp-3 with one spinule on outer proximal margin.P2 (Fig. 17B) similar to that of N. loweae sp.nov.except for intercoxal sclerite with spinules on anterior surface; coxa unornamented; basis with setules along inner margin; inner margin of exp-3 unornamented; spinules along outer margin of exp-2, 3 weaker than in N. loweae sp.nov.; inner seta of enp-3 unipinnate at distal half and stronger than in N. loweae sp.nov.
P3 (Fig. 17C) similar to that of N. loweae sp.nov.except for intercoxal sclerite with spinules on anterior surface; basis with setules along inner margin; Innermost seta of enp-3 unipinnate at distal half and stronger than in N. loweae sp.nov.; inner apical seta naked and as long as outer spine.
P4 (Fig. 17D) similar to that of N. loweae sp.nov.except for spinulose row near the base of endopod weakly developed; exp-1 with two fine setules along inner margin; middle inner seta of exp-3, longest and stronger than that of N. loweae sp.nov.; innermost seta of enp-3 stronger than in N. loweae sp.nov.; subdistal inner seta of enp-3 shorter than in N. loweae sp.nov.P5 (Fig. 16F) similar to that of N. loweae sp.nov.except for baseoendopod with four bipinnate setae, the outermost seta short and ~ 1/2 as long as the other setae.
Female.Unknown Etymology.The specific name is given in honour of Prof Dr Serdar Sak from Balıkesir University for his contribution to copepod taxonomy in Türkiye.It is a noun in the genitive case.

Discussion
The family Ameiridae ranks third within the order Harpacticoida in terms of species number (Boxshall and Halsey 2004).Members of the family inhabit a wide range of sediment types and occur in virtually all salinity regimes besides living as associates of some invertebrates (Karanovic et al. 2015).The taxonomy and phylogeny of the family Ameiridae are still problematic due to numerous ill-defined genera and lack of detailed descriptions of a great deal of species (Conroy-Dalton andHuys 1996, 1998;Karanovic et al. 2015).The genus Nitocra, which also has a notoriously challenging and problematic taxonomy, is the largest ameirid genus, at present comprising 81 valid species and subspecies (Karanovic et al. 2015;Huys 2021;Fuentes-Reinés et al. 2022).Several factors are responsible for the origin of the current taxonomic confusion surrounding the genus Nitocra.The primary issue lies in the classification of Nitocra, which encountered initial difficulties when Boeck (1865) provided an insufficient definition of the genus, offering only fragmentary descriptions devoid of illustrations.Furthermore, the majority of conventional marine genera described and outlined in the early years of the 20 th century (Sars 1907(Sars , 1911a, b, c;, b, c;Lang 1935Lang , 1936) ) did not substantially enhance the precise delineation of either the type genus Ameira or the genus Nitocra.The third factor blurring the generic boundary of the genus Nitocra is the arbitrary addition of new species, which resulted in the amalgam of phylogenetically unrelated species into the genus.Due to the taxonomic confusion within the genus Nitocra arising from these factors, resolving the complexity through a single study has become an impossible task.Consequently, urgently conducting modern standard redescriptions of numerous species within the genus with insufficient descriptions will significantly contribute to solving the problem (Karanovic and Pesce 2002).
Therefore, within this scope, the redefinition of N. affinis, one of the important polytypic species in the genus, based on lectotype material in this study has provided significant contributions to resolving the N. affinis species complex and solving the taxonomic problem within the genus.On the other hand, it is crucial to delineate species groups within the genus from a phylogenetic perspective.Despite being the largest genus within the family Ameiridae, with a notoriously difficult and problematic taxonomy, no attempts have been made to delineate species groups within the genus Nitocra until Gómez et al. (2012) who attempted to establish species groups within the genus Nitocra in order to ground the classification of the genus on a phylogenetic basis although they have not conducted a phylogenetic tree-based study.Gómez et al. (2012) recognised three species groups based on the setal formula of P1 exp-2, 3. The first group of species contained Nitocra sewelli and N. platypus bakeri which bear one inner seta on P1 exp-2 and four setae on P1 exp-3; the second group of species comprised N. reducta reducta, N. delaruei, N. blochi, N. gracilimana, N. phlegrea, and N. chelifer which lack an inner seta on P1 exp-2, but bear five setae on P1 exp-3; the third group of species accommodated the rest of the species with one inner seta on P1 exp-2 and five setae on P1 exp-3.Gómez et al. (2012) also recognised a distinct subgroup of species within the third group, based on the setal formula of P2-P4 exp-3 (7-7-7), P2-P4 enp-3 (4-5-5), and P2-P4 enp-1 (1-1-1).Nitocra affinis, N. colombiensis, N. rijekana, N. stygia, N. hamata, N. elegans, N. sonmezi sp.nov., N. loweae sp.nov., N. serdarsaki sp.nov., and N. alperi sp.nov., constitute another subgroup of species within the group which can be called the affinis group on the basis of a) by having a spinulose, long, spine-like inner middle seta on P4 exp-3, b) by having the same setal formula on the P2-P4 exp-3 (7-7-8) and P2-P4 enp-3 (4-5-5), c) by the number of seta on the P2-P4 exp-2, P2-P4 enp-1, 2 (1-1-1), d) by the elongated P1 enp-1.
Nitocra affinis is clearly distinguished from other congeners in the affinis group by the combination of the following characters in female; rostral projection reaching ~ 1/2 of the rostral length, anal operculum with 14 spinules, the reduced maxillary endopod with one slender seta, inner middle seta of P4 exp-3 strongly spinulose and long, female P5 baseoendopod with five and exopod with six setae respectively.Male P5 baseoendopod with four and exopod with six setae, respectively.Nitocra affinis has subsequently been reported from several other localities: Willey (1930) recorded it from Mangrove Lake in Bermuda, presenting only the setal formula of swimming legs and the number of spinules (fifteen) on the anal operculum.Vervoort (1964) reported N. affinis from Ifaluk Atoll in the Pacific Ocean, describing the P4 exp-3 middle inner seta as strong and long, anal operculum as spinulose, antenna exopod as one-segmented, and P1 enp-1 as slightly longer than the exopod.Unfortunately, the specific identity of the above-mentioned populations cannot be verified due to insufficient morphological data and should therefore be considered as unverified records.However, considering the isolated environments of Nitocra affinis from Bermuda and Ifaluk Atoll, it is strongly possible that each of these populations of N. affinis may represent distinct species.Rajthilak et al. (2015) recently recorded Nitocra affinis from South-east India, providing both morphological and molecular data.But, based on the morphological information provided by Rajthilak et al. (2015), even the familial ZooKeys 1191: 35-74 (2024), DOI: 10.3897/zookeys.1191.115545Nuran Özlem Yıldız & Süphan Karaytuğ: Taxonomic revision of Nitocra affinis identification of the south-east Indian population of N. affinis is uncertain and therefore this record cannot be verified.
It has been demonstrated several times that many so-called cosmopolitan harpacticoid species in fact represent species complexes (Gómez et al. 2012;Karanovic et al. 2015;George 2018;Karaytuğ et al. 2021).Vervoort (1964) and Fuentes-Reinés and Suárez-Morales (2014) proposed the potential existence of a species complex within N. affinis.Through comprehensive morphological assessments of specimens gathered from diverse geographical locations initially labelled as N. affinis, this investigation has unveiled the presence of four new species in this study, thus affirming N. affinis as a species complex.
Lack of original descriptions or insufficient taxonomic information are the main reasons for the formation of species complexes.The swimming legs segmentation and their setal formula, the number of segments in the antennule and the structure of antennary exopod are the most commonly used morphological characters for delineation of the species in harpacticoid taxonomy (Wells 2007).Therefore, in recent studies, most researchers have concentrated on finding microcharacters such as spinule ornamentation (Alper et al. 2023) or pore signature (Karanovic et al. 2015;Karanovic and Cho 2016) which proved to be helpful in differentiating closely related morphospecies.So, it was not surprising that similar results emerged in the ameirid taxonomy.For example, Karanovic and Cho (2012) distinguished two Ameira species based on setules, spinules or pore ornamentations on the somites or appendages.In this study, within the N. affinis complex four new species namely N. sonmezi sp.nov., N. serdarsaki sp.nov., N. alperi sp.nov., and N. loweae sp.nov., were revealed mostly based on micro-morphological characters which, once again, demonstrated their importance in copepod taxonomy.
Nitocra loweae sp.nov.was collected from Brighton and identified as N. affinis (Ventham 2011).Nitocra loweae sp.nov. is described on the basis on one female and a male specimen and can be easily distinguished from other congeners in the affinis group by (a) the robust, spinulose ornamentations of urosomites, (b) by the presence of twelve large dorsal spinules on the anal operculum, c) the maxilla endopod with two slender setae.Details of the specific differences are given in Table 1.Nitocra loweae sp.nov.and N. affinis can be easily differentiated by the following characters: the antennule of N. loweae sp.nov.differs from that of N. affinis in having two plumose setae and one plumose seta on the second and third segment, respectively; the two inner setae on the seventh segment set close to each other in N. affinis but widely separated in N. loweae sp.nov.; the proximal-most inner seta on the eighth segment in N. affinis is located more distally than that of N. loweae sp.nov.; N. loweae sp.nov.lacks the rostral projection on the rostrum and has five setae on the P5 baseoendopod of the male, whilst according to Gurney's (1927) description, N. affinis has four setae on the P5 baseoendopod of the male.Nitocra loweae sp.nov.has four naked distal setae and two naked lateral setae on the mandibular endopod, and two slender setae on the maxilla endopod, whereas all other species in the affinis group have four naked distal setae and one lateral seta on the mandibular endopod, and one slender seta on the maxilla endopod.In comparison to other species within the affinis group, N. loweae sp.nov. is regarded as exhibiting a more primitive state with respect to these characters.Nitocra sonmezi sp.nov.was described on the basis on one female specimen from mediolittoral zone of coast of Hatay, Turkey.Nitocra sonmezi sp.nov. is differentiated from other species of the affinis group by (a) the number of spinules on the anal operculum, (b) in the shape of P1 enp-1 which is ~ 4.3 × as long as maximum width, (c) by the ornamentation of the subdistal inner seta of P1 enp-1, (d) in the ornamentation of P5 endopodal and exopodal setae, e) in the ornamentation of urosomites and (f) in the ornamentation of the setae of P2-P4.Details of the specific differences are given in Table 1.
Nitocra alperi sp.nov.was identified as N. affinis from the Indian Ocean (Wells and Rao 1987).Nitocra alperi sp.nov.can be distinguished from other congeners of the affinis group by (a) the total length of P4 endopod segments; (b) the ornamentation of the inner seta P2-P4 enp-3; (c) the ornamentation of the inner seta of P1 enp-1, length to width ratio of this segment; (d) the surface ornamentation of somites and number of spinules on the anal operculum (seventeen) (see Table 1 for detailed comparisons).Nitocra alperi sp.nov. shares the rostral projection on its rostrum with N. affinis, N. colombiensis, and N. loweae sp.nov.
Nitocra serdarsaki sp.nov.was identified from the Aegean coast of Türkiye on the basis of the one male specimen.Nitocra serdarsaki sp.nov.can be distinguished from other new species by (a) the number of spinules on the anal operculum; (b) the ornamentation of P5 exopod and baseoendopod; (c) the ornamentation of inner setae of P2-P4 endopod-3, (d) the P1 enp-1 inner seta ornamentation; (e) the length and (f) ornamentation of the middle inner seta of P4 exp-3 (see Table 1 for detailed comparisons).Nitocra affinis has four setae on the P5 baseoendopod of the male, whereas N. serdarsaki sp.nov.has five setae on the P5 baseoendopod of the male.These new species can also be easily distinguished from N. hamata and N. elegans which are in the affinis group, by following characters; (a) shape of female P5 exopod, (b) ornamentations of the abdominal somites and (c) the ornamentations of mouthparts (Bodin 1970;Gee 2009).Nitocra hamata is distinguished from other affinis species group by the shape of female P5 exopod which is longer and slender (Bodin 1970), P1 enp-1 length and the structure of P3 enp-3.
The status of subspecific taxa within the genus Nitocra (N.reducta fluviatilis and N. sewelli husmanni) have been revised by Gómez et al (2012) to recognise them as species, based on consistent morphological differences.In this context, we have also re-evaluated the status of the subspecies of N. affinis below:

Establishment of Nitocra rijekana Petkovski, 1954
Nitocra rijekana was originally described as a form of N. affinis by Petkovski (1954) from Rijeka, Northern Adriatic (Mediterranean Sea), and has not been recorded since its original description.In the same study, Petkovski (1954) examined the material of N. affinis from Dubrovnik and compared it with that of Rijeka, determining significant morphological differences between them.The differences between Nitocra rijekana and N. affinis are as follows: (a) inner middle seta of P4 exp-3 of Nitocra rijekana is not as long as and not as strong as that of Nitocra affinis, (b) the male P5 baseoendopod with five and exopod with six setae (in Nitocra affinis 4:6).Nitocra rijekana can also be easily distinguished from its congeners by having a long but plumose inner middle seta of P4 exp-3.We believe that morphological differences between Nitocra affinis rijekana Petkovski, 1954 and Nitocra affinis are significant enough to warrant upgrading Nitocra affinis rijekana to a specific rank.The detailed comparison is provided in Table 1.Lang, 1965Lang (1965) originally described Nitocra affinis californica in a tidal pool from Monterey Bay, California.Later on, Kunz (1975) recorded Nitocra affinis californica from Gonubie, South Africa, by examining 62 specimens collected from the reef area.Kunz (1975) observed variabilities on the P5, which may indicate that Kunz (1975) was dealing with more than one species.Unfortunately, Kunz (1975) only described the P1 and P5, thus making it impossible to confirm the specific status of Kunz' (1975) specimens.After Kunz (1975) Apostolov (1980) recorded N. affinis californica from Bulgaria.Fuentes-Reinés and Suárez-Morales (2014) mentioned that the Bulgarian and South African specimens may represent different species.This observation is supported by the notably shorter P1 exopod found in both the Bulgarian and South African specimens, whose exopodal ramus extends to ~ ¾ of the length of the first endopodal segment, distinctly deviating from the characteristic of N. a. californica, where the exopod and the first endopod segment exhibit equal lengths.Lang's (1965) subspecies is here upgraded to full species rank since it differs sufficiently from Gurney's (1927) population and its congeners to warrant such status on the basis of the following characters: (a) P1 exp-3 ~ as long as exopod and exceeds the origin of P1 enp-1 inner seta (in all other species, the P1 exp-3 does not exceeds the origin of P1 enp-1 inner seta); (b) the setal ornamentation of swimming legs; (c) male P5 baseoendopod with four setae.Detailed comparison is provided in Table 1.Por, 1968Por (1968) described Nitocra affinis stygia from land-locked basins in the Red Sea.In the original description, the female P5, the abdominal segment, the caudal rami, P1, P4 exopod and male P5 were illustrated.Por's (1968) subspecies is here upgraded to full species rank since it differs sufficiently from Gurney's (1927) population and its congeners to warrant such status on the basis of the following characters: (a) large hyaline field on female P5 and its large dimensions, (b) inner middle seta of P4 exp-3 is not as long as and not as strong as in N. affinis, (c) penultimate somite surrounded by spinule (only from dorsal to lateral in N. affinis), (d) female baseoendopodal setae are almost equal in length, (e) male baseoendopod with four setae.Detailed comparison is provided in Table 1.

Establishment of Nitocra colombiensis Fuentes-Reinés & Suárez-Morales, 2014
Nitocra colombiensis is originally described as Nitocra affinis colombiensis from a lagoon in Colombia (Fuentes-Reinés and Suárez-Morales 2014).This subspecies from Colombia is here upgraded to full species rank since it differs sufficiently from Gurney's (1927) population and its congeners to warrant such status on the basis of the following characters: (a) number of setae of the male P5 endopod of; (b) number of spinules on the anal operculum; (c) shape and ornamentation of the middle inner seta P4 exp-3; (d) ratio of the P1 enp-1; (e) body ornamentation; (f) antennular setal formula; (g) maxillule basis with four setae.In the original description of Nitocra colombiensis, apical rostral projection is given as diagnostic character for the species.But the rostral projection is also observed both in N. affinis and N. alperi sp.nov.
Fuentes-Reinés and Suárez-Morales (2014) provided an identification key for affinis group.The key distinguished Nitocra colombiensis from other species with the apical rostral projection.We observed the apical rostral projection both in N. affinis and N. alperi sp.nov. in this study.Therefore, here we revised the identification key for taxa contained in the N. affinis group.
A key to the Nitocra affinis species group

Conclusion
The growing significance of microcharacters in copepod taxonomy has revealed that numerous species lacking comprehensive descriptions are, in fact, part of species complexes.In this study, Nitocra affinis was redescribed based on lectotype material which facilitated us through detailed comparison with specimens recorded and labelled as N. affinis from distantly related localities.The results clearly indicated that each of these specimens attributed to N. affinis corresponds to a distinct species.Four new species have been described from different localities, and named as N. sonmezi sp.nov., N. loweae sp.nov., N. alperi sp.nov., and N. serdarsaki sp.nov.The status of subspecific taxa of N. affinis has been re-evaluated based on the literature and four subspecies of N. affinis have been reinstated to specific rank, and named as N. stygia, N. rijekana, N. californica, and N. colombiensis.The description of the majority of the species/subspecies within the Nitocra genus is notably insufficient.While the morphological examination of mouthparts in ameirid taxa can be challenging, a detailed morphological analysis of mouthparts may significantly contribute to resolving the problematic taxonomy of the genus.Indeed, in this study, although setal formulae of the swimming legs of the four newly described species are the same as in N. affinis species, new morphological differences have been detected.For instance, there is a distinct apical extension of the rostrum of N. affinis and N. alperi.While in N. loweae, the maxilla endopod is represented by two setae of equal length, it is represented by a single seta in other species within the affinis group.These findings clearly underscore the significant contributions that detailed species descriptions will make to resolve the challenging taxonomy of the genus Nitocra.In addition to morphological studies, the phylogenetic analysis of molecular data to be obtained will provide valuable insights into both the taxonomy of the genus Nitocra and the phylogenetic relationships among genera within the family Ameiridae.

Type material. Holotype: England
The specific name is given in honour of Associate Prof Dr Serdar Sönmez from Adıyaman University for his contribution to copepod taxonomy in Türkiye.It is a noun in the genitive case.ZooKeys 1191: 35-74 (2024), DOI: 10.3897/zookeys.1191.115545Nuran Özlem Yıldız & Süphan Karaytuğ: Taxonomic revision of Nitocra affinis India • 1 ♀ (dissected on 7 slides) (NHMUK reg.no.2023.0000).Indian Ocean, Aldabra; large tide salted lagoon; W. of Point Hadroul.Coll.Pres.K.G.Mc Kenzie, 1968; J.B.J. Wells det.(material originally registered as N. affinis under NHMUK formula of swimming legs same as in N. loweae sp.nov.Male.Unknown.Etymology. Armature formula of swimming legs (not shown) same as in N. loweae sp.nov.Male.Unknown.Etymology.The specific name is given in honours of Associate Professor Dr Alp Alper from Balıkesir University for his contribution to copepod taxonomy.It is a noun in the genitive case.Drs Serdar Sak, Alp Alper, Orkan Metin Leg.This specimen was previously deposited in the collection of Biology Department of Balıkesir University which was labelled as N. affinis as a result of the faunistic project from Saros Bay, under the project number TÜBİTAK TBAG-212T105).