﻿Three new species of the sea fan genus Muricea (Cnidaria, Octocorallia, Plexauridae) from the northwest region of Mexico

﻿Abstract Twenty-one nominal species of Muricea have been reported in the Eastern Pacific with nine of them reported in the Mexican Pacific. We describe three new species of Muricea: Muriceaambaraesp. nov. and Muriceacacaosp. nov., from rocky reefs on the central and the northern Gulf of California and the Pacific coast of Baja California Sur, and Muriceamolinaisp. nov., from the Pacific coast of Baja California Sur. Muriceaambaraesp. nov. and M.cacaosp. nov. are taxonomically allied to the nominal species Muriceafruticosa Verrill, 1869 due to the morphological similarity of colony growth patterns and the phylogenetic closeness based on the mitochondrial MutS gene (mtMutS); but differ mainly in the calyx form and composition of sclerites. The main morphological differences between the new Muricea species are in their sclerite forms and color; M.ambaraesp. nov. has orange-colored colonies, thin leaf spindles and tuberculated blunt spindles, while M.cacaosp. nov. has dark brown colored colonies, strong spinous spindles and an absence of tuberculated blunt spindles. Muriceamolinaisp. nov. is phylogenetically close and morphologically similar to Muriceasquarrosa Verrill, 1869 in the growth form of the colony and tubular calyces; but has dark brown colored colonies and has calyces from the base to the branch tips. With these three new species, the total number of Muricea species reported in the Mexican northwest region increases to twelve and a total of 24 nominal species in the Eastern Pacific.


Introduction
Sea fans of the family Plexauridae of the genus Muricea Lamouroux, 1821 currently include 30 nominal species worldwide: nine species are distributed in the Western Atlantic Ocean and 21 species are distributed in the Eastern Pacific (Bayer 1959(Bayer , 1994Guzman 2015, 2016a, b;McFadden et al. 2022McFadden et al. , 2023. Guzman (2015, 2016a) subdivided the 21 Eastern Pacific Muricea species into four morphological species-groups: 1) Muricea squarrosa species-group, currently including four species characterized by the presence of tubular calyces; 2) Muricea fruticosa species-group encompassing five species with shelf-like calyces and unilateral spiny spindles; 3) Muricea plantaginea species-group including four species with a conspicuously elongated lower coenenchyme of the calyces, and foliated or thorny spindles; and 4) Muricea austera species-group encompassing eight species characterized by candelabrum-like and robust colonies, thick branches and thick coenenchyme.

Field work and morphological comparisons
All sea fan colonies analyzed for the present study were collected by scuba diving (< 50 m depth) during systematic monitoring surveys carried out between 1998 and 2020 in 250 locations in the Gulf of California and along the Pacific coast of Baja California Peninsula, Mexico ( Fig. 1A-D). The collected sea fans were preserved dry or in ethanol (96%). A portion of each sea fan colony was macerated in sodium hypochlorite to extract the sclerites, washed several times with distilled water, and preserved in 96% ethanol for further microscopic analyses. Sclerites were air-dried and attached to aluminum stubs with double adhesive bands. They were coated with gold using a sputter coater (Polaron E5100) in a gold atmosphere and observed under a Hitachi S-3000 N scanning electron microscope (SEM) at 20 kV following standard methods (Hernández et al. 2021). All digital images and image plates were edited and prepared using Adobe Photoshop 21.1.3 and Corel-PhotoImpact X3 software. Holotypes and paratypes of the three new species were described based on diagnostic internal and external morphological characters and compared with nominal species closely related within the genus Muricea (Breedy and Guzman 2015;2016 a, b). About 450 colonies of two nominal species of Muricea (M. fruticosa and M. plantaginea) and undescribed species (with specimens of the three new species) are deposited in the collection of the "Programa de Investigación para la Conservación de la Fauna Arrecifal" (PFA) of the Universidad Autónoma de Baja California Sur (UABCS), La Paz, Baja California Sur, Mexico. The external and internal morphological descriptions of the colonies of the three new species of Muricea were prepared using standard criteria and nomenclature (Bayer et al. 1983; Calvo and Breedy 2002). All holotypes and paratypes were deposited in the Invertebrate Zoology Collection, National Museum of Natural History, Smithsonian Institution, Washington, DC, USA (NMNH). Taxonomical classification was based on McFadden et al. (2022McFadden et al. ( , 2023. Maps showing the toponymy and the sampling locations of the holotypes (type locations) and paratypes of each new Muricea species were prepared using Surfer ver. 12 software (Fig. 1A-D).

Molecular analysis
Sea fan colonies preserved dry were used for molecular analyses and ten sequences (six sequences from the three new species, plus four sequences of nominal species) of a partial fragment (~931 base pair, bp) of the mtMutS gene were generated. The sequences were submitted to GenBank with accession numbers given in Suppl. material 1: data S1. In addition, twelve sequences of Muricea reported for the Eastern Pacific Ocean were downloaded from Gen-Bank (https://www.ncbi.nlm.nih.gov/genbank/) for the phylogenetic recon-struction (Suppl. material 1: data S1). All these sequences were aligned and trimmed to 493 bp to have all the sequences at the same length. For detail in the molecular analyses see the Suppl. material 1.  (Fig. 1B). The type specimens were collected by Carlos Sánchez.

Systematics
Holotype colony description. Colony with flabellate growth in one plane and laterally branched, 21 cm high and 14.2 cm wide ( Fig. 2A, Table 1). The holdfast is an irregular oval, 1.9 cm in length and 1.3 cm wide from which grows the main stem, 5.2 cm in height and 0.5 mm in diameter. There is no coenenchyme on the base of the steam lost during the collect; the base coenenchyme shows a dark gray and brownish bicolored axis. The growth of the branches is lateral and upward. Some terminal branches are short, about 5 mm in length and 3.5 mm in diameter, while the longest ones are 9.5 cm in length and 3.5 mm in diameter (Fig. 2B, Table 1). All the terminal tips are blunt and covered by calyces. Calyces in the colony range up to 1 mm in height and are 1 mm in diameter and are shelf-like in form with an imbricated arrangement. The coenenchyme color is pale yellow, but the coloration of the calyces is reddish-brown, giving the colony an overall pale and dark orange appearance ( Fig. 2A, B, Table 1).

Muricea cacao
Holotype colony description. The holotype is a brownish-red colony, growing flabellate upwards in one plane and laterally branched, 15.8 cm tall and 8.7 cm wide (Fig. 4A, C, Table 1). Polyp rows distribute imbricated (Table 1). The holdfast is 2 cm length and 1 cm wide with a cream-white color in the area without coenenchyme (lost during collect) (Fig. 4A). Two main branches arise from the base; one is 1.4 cm in length and 4 mm in width, and the other one is 1.3 cm in length and 6 mm wide. They are subdivided into three secondary branches, two of them partially fused (pseudoanastomosis) (Fig. 4A). Several terminal twigs are short (1 cm in length), with several reaching 9 cm in height. In both branches, the average diameter is 3 mm, with blunt ends (Fig. 4C). Calyces are shelflike, 1 mm in height with a diameter of 1 mm, situated all around the branches in an imbricated arrangement. Calyces are acute in the upper part of the colony or on terminal twigs and blunt at the base.
Morphological variation. All the Muricea cacao sp. nov. specimens collected and observed in situ are morphologically consistent with the macro-and micro-morphology of both the holotype (Fig. 4A-C) and paratypes with colony size range observed in situ between 2 and 55 cm height (Suppl. material 1: fig. S2E-F). The main differences among Muricea cacao sp. nov. colonies are the growing angle of the branches, the morphology likely influenced by the local water current pattern and available sea floor space on which they grow ( Fig. 8C, D). The polyps in living colonies have white neck and colorless tentacles ( Fig. 8D; Suppl. material 1: fig. S2F).
Habitat and distribution. Muricea cacao sp. nov. is present in the northern part of the Gulf of California from the Santa Rosalía region to the Midriff Archipelago Region (MAR) and along the Pacific coast of Baja California Sur, between Todos Santos and Bahía Magdalena (Fig. 1A, C). Muricea cacao sp. nov. (Fig. 1C) and Muricea ambarae sp. nov. (Fig. 1B) share similar distribution and habitat.
Remarks. Paratypes of Muricea cacao sp. nov. have low morphological variability and the main difference is the width of the branches (Suppl. material 1: fig. S4A-C). Muricea cacao sp. nov. (Fig. 4A, B) is similar to Muricea ambarae sp. nov. (Fig. 2A, B) in the colony's growth form; but differs externally because M. ambarae sp. nov. always has bicolored colonies present; reddish calyces with an orange coenenchyme and does not have pseudoanastomosis as does Muricea cacao sp. nov. In contrast, M. cacao sp. nov. has homogenous brown-reddish coloration in calyces and coenenchyme and has pseudoanastomosis (Fig. 8C, D, Table 1). The main morphological differences between both new Muricea species are in the sclerite forms present; M. ambarae sp. nov. has leaf spindles and the presence of tuberculated, bent spindles ( Fig. 3A-D), while M. cacao sp. nov. has even more strongly unilateral multi-spinous spindles, and an absence of tuberculated, bent spindles ( Fig. 5A-C, Table 1). However, due to the similarities in colony growth pattern and calyx form between M. cacao sp. nov. and M. ambarae sp. nov. (Table 1), we propose to include both species in the fruticosa species-group proposed by Breedy and Guzman (2016a). Even if Muricea cacao sp. nov. (Suppl. material 1: fig. S2E-F) is comparable with Muricea plantaginea (Suppl. material 1: fig. S2G-J), the later has prominent and elongated calyces, with elongated lower borders curved inwards (Fig. 9). Muricea plantaginea has large colonies of up to 100 cm height, simple leaf spindles and in Mexican living colonies the polyps are always orange or yellow-orange (Suppl. material 1: fig. S2G-I).
Etymology. The latinized species name "cacao" comes from the ancient prehispanic Nahuatl "cacao", which is the seed used in the making of chocolate, but in Latin is "cacao", the species name of the cocoa tree is Theobroma cacao L. This "chocolate" color is a practical diagnostic characteristic of both preserved colonies and live colonies with retracted polyps, distinguish this species in situ from other Muricea species.  (Fig. 1D). The type specimens were collected by Carlos Sánchez.
Holotype colony description. Muricea molinai sp. nov. colony coloration is gray with dark gray calyces and a creamy light gray coenenchyme (Fig. 6A, B). The colony is flabellate growing in one plane and laterally branched, reaching 15 cm in height and 14.7 cm in width ( Table 1). The holdfast is 22 mm long and 13 mm wide. Two main stems arise from the holdfast one with 2.4 cm and the second one with 4.5 cm in height, subdividing laterally in stems of 7 mm in diameter (Fig. 6A). The growth branching pattern is upward, except six secondary branches, on both sides of the colony, which display more of a downward-growing trend. Terminal branches are up to 8.7 cm tall with blunt ends of 5 mm diameter. Calyces are tubular, 3 mm in height with a 1.5 mm diameter, with an imbricated arrangement throughout the colony (Fig. 6A, B).
Holotype sclerites. The sclerites of the outer coenenchyme and calyx are unilateral (weakly) spinous spindles and tuberculated spindles (1.2-2.5 mm in length) (Figs 6C, 7A-C). The spindles are curved or straight; most of them have acute ends or are bifurcated; tuberculated spindles with blunt ends are rare. The inner coenenchymal sclerites are mostly straight or curved spindles with acute ends (0.2-1.2 mm length), but there are rare blunt spindles. Anthocodial sclerites are small tuberculated spindles (1.1-1.8 mm length) with acute ends and rods with marginal lobes and acute ends (Fig. 7D). The color of the outer coenenchymal sclerites is gray, or gray with pale amber areas in the largest ones. The inner coenenchymal and anthocodial sclerites are colorless (Fig. 6C).
Morphological variation. All twelve Muricea molinai sp. nov. colony specimens collected, including the paratypes ranging between 7 and 21 cm height ( Fig. 8E-G; Suppl. material 1: fig. S5A, B), were morphologically similar to the holotype. No detectable color variability or different colony growth pattern was observed among the examined colonies of M. molinai sp. nov. (Table 1). The length of the tubular calyces is the main morphological variability among M. molinai sp. nov. specimens, where several colonies have longer calyces (> 4 mm in length throughout the colony) than other colonies with shorter calyces (< 2 mm in length). The polyps of live colonies have white neck with translucent brown tentacles ( Fig. 8F; Suppl. material 1: fig. S6C).
Habitat and distribution. Muricea molinai sp. nov. is absent in the Gulf of California but overlaps its distribution and habitat with Muricea ambarae sp. nov. and Muricea cacao sp. nov. along the southwest coast of the Baja California Peninsula (Fig. 1B-D). Muricea molinai sp. nov. was collected between Bahía Magdalena and Cabo San Lucas; but it was absent at the Punta Abreojos site (Fig. 1D). Remarks. Live colonies of M. molinai sp. nov. (Fig. 8E-G) and Muricea squarrosa (Breedy and Guzman 2015) (Suppl. material 1: fig. S6A-M) are similar, but the shape of the calyces and the differences in the diameter of the branches are the most evident diagnostic feature to distinguish them. Muricea molinai sp. nov. has a distinctive colony coloration, being light gray in the coenenchyme, and dark gray almost black in the calyces which extend sparsely from the base to the tip of the branches (Fig. 8E-  It is proposed that Muricea molinai sp. nov. be included in the Muricea squarrosa species-group previously erected by Breedy and Guzman (2015).
Etymology. Muricea molinai sp. nov. is named in memory of Dr. José Mario Molina Pasquel y Henríquez , the first Mexican in 1995 to win the Nobel Prize in Chemistry. Molina played a vital role in the discovery of the Antarctic ozone hole demonstrating that chlorofluorocarbon gases were the cause of the deterioration of the ozone layer.

Molecular analysis
For several sea fan genera, the mtMutS gene is conserved to species level; but it is divergent enough to discriminate among genera and species-groups in octocorals. The partial fragment used for the phylogenetic reconstruction was highly conserved in Muricea, nonetheless and even with a lack of strong node support, Muricea ambarae sp. nov. and Muricea cacao sp. nov. were separated in a well-defined clade together with the nominal species Muricea fruticosa. In the case of Muricea molinai sp. nov., although not falling into a clade and showing unresolved relationships, it is clearly distinct from these last two new species and close to the nominal species Muricea squarrosa and Muricea hebes. The comparison of these mtMutS sequences with M. plantaginea, M. californica and M. squarrosa support the morphological evidence that, M. ambarae sp. nov., M. cacao sp. nov. and M. molinai sp. nov. are new species (Fig. 10; Suppl. material 1: table S1).

Discussion
Sea fan species assemblages have a clear latitudinal regionalization in the Gulf of California (Ulate et al. 2016). The southern region has environmental conditions influenced by the tropical surface water mass typical of the Mexican Province (Brusca and Wallerstein 1979;Hasting 2000;Portela et al. 2016). This southern region has the highest species richness and density of sea fan species of the genera Pacifigorgia Bayer, 1851and Leptogorgia Milne Edwards, 1857(Ulate et al. 2016. The Midriff Archipelago Region (MAR) (Fig. 1A) is the coldest region in the Gulf of California due to intense tidal currents and topographic upwelling (Lavín and Marinone 2003;López et al. 2006;Ulate et al. 2016), where sea fan species richness and abundance are numerically dominated by M. austera, M. fruticosa and M. plantaginea. Muricea ambarae sp. nov. and M. cacao sp. nov. share their habitat (5-20 m depth) in this region (Fig. 1B, C), where M. plantaginea is the most abundant species. We show SEM morphological evidence that the three new species have a distinct calyx and terminal twig morphology that differs from M. plantaginea (Fig. 9). Muricea ambarae sp. nov. and M. cacao sp. nov. were first reported in the MAR as undescribed species labeled as: Muricea sp. 2 and Muricea sp. 5 in Ulate et al. (2016). Muricea austera, M. fruticosa and M. plantaginea are also numerically dominant at > 40 m seafloor depth associated with low temperatures in the southwestern region of the Gulf of California between Loreto and Cabo San Lucas. However, M. ambarae sp. nov. and M. cacao sp. nov. have not been observed in the southern region of the Gulf of California (Ulate et al. 2016); they are also distributed along the Pacific coast of the Baja California Peninsula, Mexico, between Cabo San Lucas and Punta Abreojos, Baja California Sur, Mexico (Fig. 1B, C). Muricea molinai sp. nov. is distributed in shallow waters (< 20 m) in the Bahía Magdalena region, but it can be observed deeper >50 m in Cabo San Lucas (Fig. 1D). Bahía Magdalena is located at a transitional biogeographic region between the Mexican Province and the California Current Province with seasonal cold waters (Hasting 2000), which could explain the preference of Muricea species for temperate habitats.
The molecular analysis of species of the genus Muricea and other sea fan genera distributed in the Eastern Pacific is still incipient (Herrera et al. 2010;Gómez 2012;Vargas et al. 2014;Poliseno et al. 2017 (Herrera et al. 2010;Poliseno et al. 2017) and several other Muricea spp. (Wirshing et al. 2005;Vargas et al. 2014;Ament-Velásquez et al. 2016) (Suppl. material 1: table S1). Few studies have tried so far to infer the phylogenetic relationships of species of the genera Muricea with single mitochondrial genes or complete mitogenomes. Therefore, there is still no comprehensive knowledge for taxonomic species delimitation for most Muricea species distributed in the Eastern Pacific (Vargas et al. 2014;Poliseno et al. 2017). This lack of morphological and genetic knowledge is mainly the reason that  Muricea has historical taxonomic problems in species identification of specimens observed in the field and from preserved material (Breedy and Guzman 2015;2016a, b;Ulate et al. 2016) as well as the lack of systematic benthic surveys to cover most of the rocky and pebbled sea floor habitats in the northwest Mexico region (Hernández et al. 2021). Despite these historical limitations, our molecular evidence (mtMutS gene) supports the conclusion that the three species discovered in the present study are indeed new species compared with other nominal species distributed in the region of study (Figs 1, 10). Breedy and Guzman (2015) redescribed the genus Eumuricea Verrill, 1869 in the Eastern Pacific and reclassified it within Muricea, proposing that it be included in the Muricea squarrosa species-group, which includes species with tubular calyces. We propose to include Muricea molinai sp. nov. in the M. squarrosa species-group suggesting that M. molinai sp. nov. and M. squarrosa have anti-tropical distribution inhabiting subtropical latitudes. Anti-tropical species are populations with a disjunct distribution, that is, the ancestor originated on one side of the tropics, dispersing later and occupying geographical areas in the opposite hemisphere, being absent within the rocky Mexican Province and Central American gap, as has been observed in reef fish species assemblages (Hasting 2000). Later, Breedy and Guzman (2016a) redescribed several Muricea species, proposing the Muricea austera species-group, the M. fruticosa species-group (where we included Muricea ambarae sp. nov. and Muricea cacao sp. nov.), and the M. plantaginea species-group as mentioned earlier. The practical purposes of these Muricea species-groups (which do not necessarily have a phylogenetic relationship) and the precise morphological descriptions of these nominal species have facilitated species identification in field ecological studies, allowing the description of new species such as the most recently described species Muricea subtilis Guzman, 2016 (Breedy andGuzman 2016b). Although the dominant octocoral genera in the Eastern Tropical Pacific are Leptogorgia, Muricea, and Pacifigorgia (Breedy and Cortés 2015;Abad et al. 2022), it is still difficult to infer the gamma species richness in the Northern Mexican Pacific because several new species still await formal description. Three species of Leptogorgia were recently discovered in the Gulf of California (Hernández et al. 2021). The discovery of these three frequent and abundant new Muricea species reported in the present study is due to the previous lack of research effort carried out in both the Midriff Archipelago Region and the Bahía Magdalena region (Fig. 1A), and overall, the historical lack of taxonomic knowledge of Muricea species in the northwest region of Mexico.