Kumanoa mahlacensis ( Batrachospermales , Rhodophyta ) in a Mediterranean coastal wetland , a new species for the European continental algal flora

García-Fernández, M.-E., Vis, M.L. & Aboal, M. 2015. Kumanoa mahlacensis (Batrachospermales, Rhodophyta) in a Mediterranean coastal wetland, a new species for the European continental algal flora. Anales Jard. Bot. Madrid 72(1): e018 Morphological characters and molecular sequence data of the plastidencoded RUBISCO large subunit gen (rbcL) confirmed the first record of Kumanoa mahlacensis (S. Kumano & W.A. Bowden-Kerby) Entwisle, M.L. Vis, W.B. Chiasson, Necchi & A.R. Sherwood in Europe from a Spanish Mediterranean coastal wetland. The genus Kumanoa has a worldwide distribution and inhabits in both lotic and lentic habitats, but information about the ecological requirements of species is scarce. In this study, the morphology and ecology of Spanish material is described in order to gain a broader insight of the distribution of species from Mediterranean

Kumanoa mahlacensis was originally included in Batra chospermum section Contorta as Batrachospermum mahla cense (Kumano & Bowden-Kerby, 1986).It is characterized by primary fascicles up to 9 cell-storeys, numerous secondary fascicles, asymmetric carpogonium, carpogonial branches curved, twisted or tightly coiled, with 5-15 barrel-shaped cells clearly different from the fascicles cells and 1-2 hemispheric carposporophytes up to 170 μm in diameter (Kumano, 2002).It is distinguished from closely related species by having dense carposporophytes lower than the whorl, abundant secondary fascicles and smaller carposporangia (Necchi & Vis, 2012).This taxon was first described from a river in Guam, a Pacific island with tropical climate, on volcanic terrains, with an average precipitation of 2.000 mm per year (Kumano & Bowden-Kerby, 1986).Later, it was reported from shallow streams in a mixed deciduous forest of the tropical area of Tambol Huay Kha Yeng, Amphur Thong Pha Phum, Thailand (Traichaiyaporn & al., 2004), and in two dry areas of the southwestern USA, Upper Tonsil Lake Carlsbad Springs Park, New Mexico and West Big Prong Creek, Texas (Vis & al., 2012).
In this study, Kumanoa mahlacensis is reported for the first time in Europe, the morphological and ecological characteristics of the Spanish material are compared with the literature and sequence data are provided to confirm the identity.

Study area
The specimens were collected in an irrigation ditch from Pego-Oliva Natural Park located between the towns of Pego and Oliva (Valencia-Alicante, Spain) (Figs. 1, 2a).Pego-Oliva Natural Park is one of the most important wetlands on the Spanish Mediterranean coast with about 14 km 2 fed by groundwater.The average annual rainfall and temperature is 905 mm and 18 °C, respectively (Viñals & al., 1990).These climatological characteristics explain the subtropical character of the vascular flora (Boira, 1988;Carretero, 1990).The high salinity gradient from freshwater to brackish and the various levels of nutrient concentrations also contribute to the floristic richness and algal biodiversity of the area.

Sampling and processing
The specimens were collected during extensive seasonal sampling of different habitats of the wetland: rivers, sources, irrigation ditches, rice fields and salt water sources in the Natural Park.Water temperature, pH, conductivity, dissolved oxygen, light intensity and current velocity were measured in situ with portable equipment.At the same time, water samples were collected, filtered (Millipore 0.45 μm) and frozen with liquid nitrogen in the field and kept at −20 °C or refrigerated in the dark until further analysis in the laboratory.The water chemistry was analyzed (NO 3 − NO 2 − , PO 4 3− and Si) spectrophotometrically with MERCK kits (Spectroquant © ).The specimens were examined and measured with a stereomicroscope and a Leica DMRB microscope equipped with a computer program to scan images (Spot RT Software v3.0.).The morphological vouchers were included in the MUB Herbarium of the University of Murcia (5152 MUB-ALGAE).

Molecular data analysis
Fresh thalli were desiccated in silica gel and kept frozen at −20 °C.These samples were ground in liquid nitrogen, and DNA was extracted using the (NucleoSpin ™ Plant (Clontech Laboratories Inc., Mountain View, CA, USA) following the  manufacturer's protocol.Double-stranded PCR amplification was performed in an Applied Biosystems 2720 thermocycler (Applied Biosystems [ABI], Foster City, CA, USA) as previously described (Entwisle & al., 2009).Polymerase chain reaction for rbcL for a total volume of 100 µL consisting of 2 µL genomic DNA; 20 µM each of dATP, dTTP, dGTP and dCTP; 0.4 µM of each primer; 2 mM MgCl 2 ; and 10× reaction buffer with 1 unit of Taq Polymerase (Invitrogen, Carlsbad, CA, USA) (Entwisle & al., 2009).A portion of the large subunit of ribulose 1,5-biphosphate carboxylase/oxygenase (rbcL) gene was amplified using primer pair F160 and rbcLR (Stewart & Vis, 2007).The PCR product was purified using UltraClean ™ PCR Clean-up (Mo Bio Laboratories, Carlsbad, USA) according to the manufacturer's protocol.The PCR product was sequenced using the ABI PRISM ™ Big Dye Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems (ABI), Foster City, CA, USA) and an ABI 3100 Genetic Analyzer.

Distribution and biogeography
In order to study the distribution and biogeography of Kumanoa mahlacensis, references from both the scientific literature and databases (Genbank) were compiled.This data set was overlapped in ArcMap (ESRI), with updated Köppen-Geiger climate classification maps provided by the Food and Agriculture Organization of the United Nations (1999).
Morphometric parameters of Spanish specimens were similar to those reported for specimens from Guam (Kumano & Bowden-Kerby, 1986) and Thailand (Traichaiyaporn & al., 2004) (Table 1).The specimens from Spain had slightly smaller whorl and carposporophyte diameters, and length of carpogonium than those from Guam and Thailand.All the diagnostic features and quantitative characteristics measured are within those given for the species in Necchi & Vis (2012), except that this specimen extends the lower range of whorl diameter from 250 µm to 220 µm.

Environmental and ecological characteristics
Specimens were collected in an irrigation ditch only in September with a water temperature of 20 °C, a pH of 7.5 and a conductivity of 610 µS/cm (Table 2).The same locality was visited in three other occasions during year 2009, but no gametophytes were observed.The annual average water temperature was ~20 °C, the mean annual pH varied slightly ~7, dissolved oxygen remained around 8 mg/L and the incident radiation was relatively high.The nitrate concentration was high during the entire year with the maximum concentration in November and minimum in August.Nitrite, orthophosphate and silicate concentrations were low and remain fairly constant throughout the year.
The conductivity, nitrate and nitrite concentrations were higher in the Spanish wetland than in Thailand, but orthophosphate concentration was lower (Table 2).However, the temperature and the pH were very similar in all locations with measurements.In general, this species seems to have a wide ecological range: from irrigation ditches with high nutrient concentration to clean and shallow freshwater streams with slow flow (Kumano & Bowden-Kerby, 1986;Traichaiyaporn & al., 2004).

Molecular data
The sequence data of a 1282bp region of the rbcL gene (Genbank number JQ713334) confirmed that the specimens collected in Pego-Oliva Natural Park belong to Kumanoa mahlacensis.This specimen had 100% sequence identity with the two Genbank sequences (JN589006, JN589007) from New Mexico and Texas, respectively (Vis & al., 2012).There are no sequence data for the type location in Guam or the report from Thailand (Kumano & Bowden-Kerby, 1986;Traichaiyaporn & al., 2004).

Distribution and biogeography
Kumanoa mahlacensis has been reported from only five sites worldwide: Guam (Pacific Ocean), Thailand (Asia), New Mexico and Texas in the southwest US (North America), and in Pego-Oliva Natural Park, Spain (Europe) (Fig. 3).
According to Köppen-Geiger climatic zone, the species has been documented in 3 of 5 climatic regions, from Tropical regions (Kumano & Boden-Kerby, 1986;Traichaiyaporn & al., 2004) to Arid (Vis & al., 2012) and Temperate regions, and in 4 of the 31 sub-climatic classes (Equatorial, Monsoon, Mediterranean and Desert), which were defined by temperature and precipitation conditions (Kottek & al., 2006).The area where the specimens were collected in Spain is included in the Mediterranean thermoclimate with a subhumid ombroclimate (Rivas-Martínez, 1987).
Until recently, K. mahlacensis was considered a tropical species (Kumano & Boden-Kerby, 1986;Traichaiyaporn & al., 2004), but it probably has a cosmopolitan distribution.A similar case occurred with Nostochopsis formerly considered a tropical genus, which also has a cosmopolitan distribution (Moreno & al., 2012).Although Mediterranean wetlands are important refugees for biodiversity and are included in several International Conservation Treaties (Ramsar Convention  Secratariat, 2011), the algal communities have been very poorly studied (Uhel & al., 2010) and most likely other overlooked species will be discovered in future (Cantoral-Uriza & Aboal, 2001;García-Fernández & al., 2011).This work represents a new contribution to this effort.

Fig. 1 .
Fig. 1.Location of Pego-Oliva Natural Park (E Spain): a, in Valencia Autonomous Community; b, between the provinces of Valencia and Alicante; c, the sampling point is enclosed by a circle.

Fig. 3 .
Fig. 3. World distribution of Kumanoa mahlacensis and climatic regions (Köppen-Geiger classification system).Triangles represent records with genetic data and circles are records without these data.

Table 1 .
Morphological comparison of Kumanoa mahlacensis from the irrigation ditch of Pego-Oliva Natural Park with the literature