A new species of Bestiolina (Crustacea, Copepoda, Calanoida, Paracalanidae) from coastal waters of the Colombian Pacific, including a worldwide key for the identification of the species

Abstract Plankton samples obtained from estuarine waters of the Colombian Pacific yielded adults specimens of an undescribed species of a paracalanid copepod of the genus Bestiolina. It most closely resembles two Asian species; B.sinica (Shen & Lee, 1966) from China and B.arabica (Ali, Al-Yamani & Prusova, 2007) from the Arabian Gulf. These three species share the absence of spinules on the posterior surfaces of exopod segments of legs 2, 3 and 4. Bestiolinasarae Dorado-Roncancio & Gaviria, sp. n. can be easily separated from B.sinica by the number of spinules on the anterior surface of endopod 2 of legs 2 and 3, and by the absence of spinules on the posterior surface of second endopod of leg 4. It can be distinguished from B.arabica by the presence of spinules on the posterior surface of endopod 2 of same legs (absent in B.arabica), and the size of spinules on the anterior surface of the same segments. The only other species known from the Americas, B.mexicana (Suárez-Morales & Almeyda-Artigas, 2016), can be distinguished from Bestiolinasarae Dorado-Roncancio & Gaviria, sp. n. by the presence of spinules on the posterior surface of the leg 2 first exopodal segment and the morphology of the mandible blade. The morphological and meristic differences to the eight known species of the genus are presented. An identification key to the species of Bestiolina is provided.


Introduction
The family Paracalanidae is represented by seven genera (Razouls et al. 2018;Walter and Boxshall 2018) and are among the common estuarine and coastal planktonic copepods of tropical and subtropical latitudes (Suárez-Morales and Almeyda-Artigas 2016). The paracalanid genus Bestiolina (Andronov 1991) currently includes eight species and was originally named Bestiola (Andronov 1972). It was subsequently renamed because Bestiola was preoccupied by an insect generic name (Nikolskaya 1963). It can be considered as a relatively recently described genus in relation to the first descriptions of marine planktonic copepod species done at the middle of the 19 th century (i.e., Dana 1852;Claus 1863;Boeck 1865;Brady 1899). Most species of Bestiolina were described in the last 20 years (Mulyadi 2004;Ali et al. 2007;Moon et al. 2010; Suárez-Morales and Almeyda-Artigas 2016). The poor knowledge of the diversity and distributional patterns of the genus could be explained by their small size (670-1008 µm), inappropriate sampling techniques (nets with mesh size > 200 µm) and confusion with copepodite stages of other paracalanid species. The lack of information about Bestiolina in the tropical eastern Pacific could also be explained by the few faunal surveys done in coastal waters of the region.
Bestiolina can be characterized as a coastal-neritic copepod genus that lives in shallow waters near the coastal areas (Bradford-Grieve 1994;Boxshall and Hasley 2004). Species of Bestiolina are concentrated in tropical latitudes of different oceans, and its origin has been speculated to be Indo-Malayan (Ali et al. 2007). Except for the record of Bestiolina mexicana in the Gulf of Mexico (Suárez-Morales and Almeyda-Artigas 2016), no other species of Bestiolina have been hitherto recorded in coastal waters of the Americas.
During the development of a project to evaluate marine bioinvasions in the Colombian Pacific and their relation with marine traffic, zooplankton samples were collected in three different coastal areas. Specimens of Bestiolina present in several samples could not be assigned to any known species of the genus and was thus deemed as new. Based on several adult female and male specimens available, the species is described and illustrated herein.

Methods
Zooplankton samples were collected only once in six localities from three major port areas of the Colombian Pacific coast (Fig. 1)  Samples were obtained via surface trawls using a standard zooplankton net with 150 µm mesh size hauled for 2 minutes from a boat travelling at approximately 2 knots. Zooplankton was narcotized with MgCl 2 (Suther and Rissik 2009) prior to fixation and preservation in ethanol 80% at a 1:3 ratio.
Dissection techniques followed Björnberg (1981). Specimens were dissected in glycerine using sharpened tungsten needles. Specimens were then mounted in Entellan (J. Dorado) and lactophenol (S. Gaviria) and sealed with varnish. Animals were studied using a Zeiss Ax10 Scope A1 (J. Dorado) and a Nikon Ellypse 200 (S. Gaviria). Drawings were performed based on images obtained with a Zeiss Ax10 Scope equipped with a digital camera.
Type specimens were deposited at the Museo de Historia Natural Marina de Colombia, Santa Marta, Colombia (MAKURIWA) of the Instituto de Investigaciones Marinas y Costeras INVEMAR, and at the Naturhistorisches Museum Wien (NHMW) in Vienna, Austria.
Environmental parameters were measured in situ with a multiparametric probe (Hach-HQ40d) and water transparency was determined with a Secchi disk. At each site, 200 ml water was filtered through glass fibre filters (Whatman GFC) for chlorophyll-a analysis (ex situ using spectrophotometry). Water temperature, salinity, dissolved oxygen, Secchi depth and chlorophyll-a data, together with standard deviation (SD) were as follows: surface water temperature x -= 28.   Etymology. The new species is named in honour of Sara Dorado, an important member of the family of the first author, who passed away one year before the discovery of the species. The name of the species is a feminine noun in genitive singular.

Taxonomy
Type locality. Near Buenaventura harbor (03°53'49.054"N; 077°03'44.3"W) ( Fig. 1), Eastern Pacific Ocean, Colombia. At the type locality, the waters are characterized as coastal and estuarine. The type locality belongs to the Buenaventura natural ecoregion of the Colombian Pacific according to the classification of Diaz and Acero (2003). The area is characterized by bays, with an average depth between 12 m and 15 m, and tectonic estuaries, which include a wide variety of habitats such as sandy and rocky beaches, mudflats, large areas of high-productivity mangroves, sandstone cliffs and soft-sediment floodplains. Many rivers and streams empty into the sea, bringing high amounts of sediments and causing variations in the physical and chemical conditions of the waters (Lazarus-Agudelo et al. 2007, Betancourt andPortela et al. 2011). Precipitation in the region is very high (> 5000 mm/y) (Dimar 2002). Water chemistry can be characterized as follows: surface temperature ranges between 26.6 °C and 29.7 °C; salinity between 1.3 and 30 psu; relative humidity close to 90%. Precipitation for the area ranges between 5000-7000 mm per year, semidiurnal tides with an average range of 4.1 m (Cantera and Blanco 2001).
Differential diagnosis. Bestiolina of small size (female 0.64-0.73 mm, male 0.63-0.75 mm), with body divided in prosome and slender urosome. Cephalic dorsal hump present in male. Rostrum short and stout divided in acute points. First pedigerous somite fused with cephalothorax, fifth pedigerous somite separated from preceding somite. Posterolateral margins of fifth pedigerous somite rounded and ornamented with small spinules. Genital double-somite with ventral protuberance in adult females. Exopods of legs 2-4 with anterior and posterior surfaces of all segments without spinules. Endopod 2 of legs 2 and 3 with anterior surface mostly with 3 small spinules and posterior surface mostly with 4 large spinules. Leg 5 of female rudimentary, unsegmented, consisting of a pair of rounded lobes, lobes with smooth margin. Leg 5 of male asymmetrical, right leg as in female, left leg long, 5-segmented, last segment with long distal spine.
Description of holotype female. ( Fig. 2A) Length of specimen measured from tip of rostrum to posterior margin of caudal rami: 0.70 mm. Body robust, widest section at second somite, anterior part of cephalosome rounded. Rostrum short and stout, divided into acute points (Fig. 2B). First pedigerous somite completely fused with cephalosome. Second, thirth and fourth pedigerous somites free. Fifth pedigerous somite completely separated from fourth, with posterolateral margins rounded and bearing small spinules (Fig. 2C).
The maxillule, maxilla and maxilliped are described according to Ferrari and Ivanenko (2008).
Leg 1 (Fig. 4A): coxa with row of short setae on inner margin, and 2 setae on outer margin. Basis with 1 seta on inner margin. Exopod 3-segmented; first segment with 1 spine distally on outer margin, inner margin with 1 seta; second segment, outer margin naked, inner margin with 1 seta; third segment outer margin with 2 setae, distal margin with 1 seta, inner margin with 4 setae. Endopod 2-segmented; first segment, inner margin with 1 seta; second segment, outer margin with 1 seta, distal margin and inner margins each with 2 setae. Anterior and posterior surfaces of all segments without spinules.
First to fifth pedigerous somites and swimming legs like in female. Urosome 5-segmented. Leg 5 (Fig. 5B) typical for the family, right leg consisting of a rounded lobe as in female, left leg elongate, 5-segmented, distal segment with apical spine.
Leg 3, endopod 2: variability of the ornamentation pattern was also noted on this leg but less accentuated than in leg 2 (Table 1). Anterior surface with 3 short spinules on anterior surface and 4 long spinules on posterior surface (holotype and paratype NHMW 26310) on both left and right legs; one paratype (MAKURIWA INV-CRU8993) shows 1 additional spinule on both legs on anterior and posterior surfaces (4+5 instead of 3+4). One paratype (NHMW 26309) shows 1 additional spinule on posterior surface (total 3+5) of left leg. In general, the most common spinulation pattern of second endopods in legs 2 and 3 is 3 spinules on anterior surface and 4 on posterior surface. Males (n = 3) show variability on body length x -= 0.70 ± 0.06 (0.63-0.75 mm). No variability was noted on spinulation pattern of the 3 studied specimens.

Discussion
Specimens from the Colombian Pacific were identified as belonging to the genus Bestiolina based on the diagnostic characters of the genus (Bradford-Grieve 1994): relatively short rostrum, presence of one seta on the inner margin of basis of leg 1, outer margin of exopo- dal segments 2 and 3 of legs 2-4 without teeth, and distal segment of endopods of legs 3 and 4 with 6 setae. The typically reduced female fifth leg, with a bilobated form in female, the dorsal hump of cephalothorax and the asymmetrical legs 5 with long left leg and bilobated right leg in male, constitute the most discriminative characteristics of Bestiolina.
Adult members of genus Bestiolina can be confused with juvenile stages of other Paracalanidae due to the size of the anal segment (slightly longer than urosomites 2 and 3 together). Copepodites V of the other Paracalanidae have the same pattern and only the adult stages show an anal segment as long as urosomite 3. Additionally, the morphology of female leg 5 in immature stages of Acrocalanus and Parvocalanus is similar to adult stages of females of Bestiolina.
Specimens of Colombian Bestiolina were compared with the eight known species of the genus (Tables 2, 3).
In Bestiolina, the ornamentation of endopods and exopods of legs 2-4 is important to distinguish the species (Table 3). Bestiolina sarae sp. n. shares with B. sinica and B. arabica the absence of spinules on anterior and posterior surfaces on exopod segments of legs 2-4, but females of B. sinica are longer ( x -= 0.94 mm) than Bestiolina sarae sp. n. ( x -= 0.70 mm, Table 2), and have 4 and 5 spinules on the anterior surface of endopod 2 of legs 2 and 3, respectively (vs 3 and 3, respectively, in Bestiolina sarae sp. n.; Table 3). The new species also shares with B. arabica the number of spinules (3) on the anterior surface of endopod 2 of legs 2 and 3, but this species lacks spinules on the posterior surface of the same segments (vs 4 in Bestiolina sarae sp. n.). Additionally, spinules of the anterior surface of this segment in B. arabica are large while those of Bestiolina sarae sp. n. of the same surface are small.
The most relevant character to distinguish species of Bestiolina is the spinulation pattern on the anterior and posterior surfaces of endopod 2 of legs 2 and 3. B. sarae sp. n. bears 3 (anterior surface) and 4 (posterior surface) spinules, where as all other species have a different combination pattern: 4+3 in B. coreana, 0+5 in B. similis, 0+4 (leg 2) and 0+5 (leg 3) in B. amoyensis, 3+0 in B. arabica, 4+4 (leg 2) and 4+5 (leg 3) in B. sinica, 4+0 (leg 2) and 4+3 (leg 3) in B. zeylonica, 2+4 (leg 2) and 2+0 (leg 3) in Differences with B. mexicana are the form of the rostrum, which is short and stout in Bestiolina sarae sp. n. and long and with slender filaments in B. mexicana, and the morphology of the cutting edge of the mandible (two dorsal teeth in B. mexicana, one in Bestiolina sarae sp. n.). Additionally, B. mexicana bears spinules on the posterior surface of first exopod segment of leg 2, while this surface is naked in Bestiolina sarae sp. n. (Table 2).
Although a high variability on the spinulation pattern of the endopod 2 of legs 2 and 3 was observed in Bestiolina sarae sp. n. (Table 1), sometimes also differing between right and left legs of the same individual, the most common pattern is represented by 3+4 (three small spinules on the anterior surface and four large spinules on the posterior surface). Other spinulation patterns observed on the same segments are: leg 2 (4+4, 4+5) and leg 3 (3+5, 4+5). In contrast, B. arabica with leg 2 (3+0) and leg 3 (3+0) does not show this pattern. Although the 4+4 pattern (typical in B. sinica) was observed in endopod of leg 2 in two specimens of Bestiolina sarae sp. n. (left and right leg 2 in paratype NHMW 26309; right leg 2 in paratype 26310), B. sinica bears four spinules on the posterior surface of endopod 2 of leg 4, while in Bestiolina sarae sp. n. it is always naked.
Bestiolina sarae sp. n. is a component of plankton of tropical waters (28.7-28.8 °C). It was found in brackish waters with low salinity (23.0-23.9 pt), dissolved oxygen from 6.4 to 6.7 mg/L, and primary productivity (in terms of chorophyll-a) with 2.8-3.0 µg/l. Where B. sarae was collected, light penetration of the water was low (Secchi disk depth 3.6-4.8 m).
In the present study, B. sarae sp. n. showed a wide range of densities, from 3 to 624 individuals/m 3 . As the species was found in all six localities, separated by up to 500 km from each other, it seems to be widely distributed in the area and could represent a typical copepod of the zooplankton of the Panama Bight. Due to the climatological and oceanographical characteristics of the study area (Dimar 2002;Fernández-Álamo and Färber-Lorda 2006), it seems likely that this species also occurs in coastal waters of the Baudó and Sanquianga ecoregions of the Colombian Pacific (Diaz and Acero 2003) and in other countries of the Eastern Tropical Pacific, such as Panama and Ecuador. It is conceivable that B. sarae also occurs in other regions influenced by the El Niño Southern Oscillation phenomenon as result of the tropicalization of species (Carrasco and Santander 1987).

Conclusions
With the discovery of Bestiolina sarae sp. n., the number of species of the genus is increased to nine, with two of them living in coastal waters of the tropical Americas. It is the first representative of the genus in the Eastern Tropical Pacific and seems to be native to the Panama Bight. It seems possible that the species is also distributed in neighbouring coastal waters such as those of Ecuador and Panama, and it might also be expected in other areas influenced by the climatological and oceanographical El Niño Southern Oscillation.
Bestiolina sarae sp. n. was probably not detected in the past due to the paucity of surveys in the study area, the use of inappropriately sized zooplankton nets, and the confusion of adults with juvenile stages of other paracalanids. For future studies, we recommend the use of nets with mesh sizes less than 150 µm, which will allow for the collection of small copepods such as B. sarae sp. n. and other members of Paracalanidae.