Marine Macroalgal Reference Culture Collection at the University of the Philippines Marine Science Institute (UP-MMARCC): Status and prospects for advancing Philippine phycology

(1)

Volume 16 Issue 1 - 2022 | 17

Seaweeds research in the Philippines—from studies on their diversity, natural products chemistry, and the utilization of their derivatives—is largely based on spot collections of large and conspicuous components of the seaweed flora found along the coasts. Such efforts are often focused on commercially important seaweeds; thus, most of the smaller and even microscopic seaweeds remain understudied, if not completely overlooked. Consequently, little to none is known on many aspects of the biology, ecology, and even biochemistry of these components of the Philippine seaweed flora. To understand aspects of seaweed biology and serve as a facility for preserving the genetic resources of Philippine seaweeds, we established the Marine Macroalgal Reference Culture Collection at the University of the Philippines Marine Science Institute (UP- MMARCC). We are currently maintaining 446 seaweed strains (or isolates) collected from several coastal and offshore areas in the Philippines, the latter including the Kalayaan Island Group in the West Philippine Sea, and 25 isolates from Okinawa, Japan. To our knowledge, the UP-MMARCC is the most diverse and widely sampled culture collection in the Philippines so far. Moreover, our preliminary molecular-assisted biodiversity studies suggest that UP-MMARCC houses several isolates that are either new records to the Philippines or putative new taxa. We anticipate that with continued support, we will be able to sustain and expand our culture collection, not only to facilitate discoveries but also to cater to the needs of the Philippine seaweed industry and in support of its call for diversifying our seaweed commodities and their products..

Keywords: germling emergence method, marine biodiversity, seaweed culture, Pacific Ocean, tropical seaweeds, rbcL

**Wilfred John E. Santiañez*, Christian Ace T. Guerta, and John Michael L. Lastimoso**

Marine Macroalgal Reference Culture Collection at the University of the Philippines Marine Science Institute (UP-MMARCC): Status and prospects for advancing Philippine phycology

G.T. Velasquez Phycological Herbarium and The Marine Science Institute, College of Science, University of the Philippines, Diliman, Quezon City, Philippines

*Corresponding author: [email protected]; (02) 8922- 3958

Date Submitted: 12 July 2022 Date Accepted: 27 October 2022 Introduction

Marine biodiversity knowledge in the Philippines remains patchy and the inventory of our marine resources is often limited to charismatic and/or economically important species. Despite being ecologically and economically important, marine macroalgae (seaweeds) are often neglected in biodiversity assessments. Seaweed inventories, when available, are based on limited assessments often focused on large and conspicuous seaweeds of shallow intertidal areas.

Meanwhile, the diversity of minute and cryptic components, including those in deeper waters, are either overlooked or understudied.

The recent account on seaweed biodiversity of the Philippines reported 1,065 seaweed taxa, the most diverse within the tropical western Pacific (Lastimoso & Santiañez 2021). This account, however, is primarily based on voucher herbarium specimens deposited at key herbaria, such as the Gregorio T. Velasquez Phycological Herbarium (MSI) of the Marine Science Institute, University of the Philippines (UPMSI) in Diliman, Quezon City. Herbaria serve as important facilities in documenting a country’s biodiversity information (Nelson et al. 2013). However, collections and

assessments on Philippine seaweeds such as those of MSI are mostly focused on relatively larger and conspicuous taxa (Trono 1999; Ganzon-Fortes 2012; Ganzon-Fortes & Santiañez 2021). These efforts tend to overlook minute and cryptic taxa or may miss accounting for other seaweeds in the area that may be occurring in their microscopic stages. Fortunately, this gap could be bridged by establishing a repository of living seaweeds called a reference culture collection.

Establishing and maintaining (algal) reference culture collections are gaining increased attention in many areas considering the economic potentials of marine algae as well as due to local and global threats to biodiversity. Specimens in a reference culture collection facility are maintained to preserve important (algal) strains (biobank), facilitate biomass production and easier validation of taxa when prospecting for bioactive compounds, and research algal life history and/

or adaptation, among others (Campbell & Lorenz 2020).

Seaweed reference culture collections have been established in several countries, mainly in universities, where active phycological research is being conducted such as in Japan (Kawai et al. 2020), the United States of America (Starr &

Zeikus 1993), and Australia (West 2005). Culture collection that houses and accounts for the diverse seaweed resources of the Philippines is presently unavailable. Although, efforts have already been made on maintaining some strains of the commercially important species Eucheuma denticulatum (N.L. Burman) Collins & Hervey and Kappaphycus spp. at the UPMSI (initiated in the late 1990s by Dr. Gavino C. Trono, Jr. and Dr. Edna T. Ganzon-Fortes) and in other institutions such as the (1) National Seaweeds Technology Development Center, Bureau of Fisheries and Aquatic Resources of the Philippine Department of Agriculture in Sorsogon, the (2) Aquaculture Department of the Southeast Asian Fisheries Development Center in Iloilo (Luhan & Sollesta 2010), and the

(2)

(3) Seaweed Research and Development Center of Mindanao State University in Tawi-Tawi, Mindanao. Indeed, efforts and studies on the culture of seaweeds in the Philippines remain limited and tend to focus on economically important seaweeds such as E. denticulatum and Kappaphycus spp. (Dawes &

Koch 1991; Dawes et al. 1993; 1994), Sargassum spp. (Aaron- Amper et al., 2020), and Halymenia durvillei Bory de St.

Vincent (Trono 2014; Santiañez et al. 2016; Rula et al. 2021).

As such, we have established a reference culture collection for the seaweed resources of the Philippines at the UPMSI—

that is, the UPMSI Marine Macroalgal Reference Culture Collection (UP-MMARCC)—to complement the herbarium- collections-based cataloging of seaweed biodiversity and in preserving seaweed genetic resources including our work on the barcoding of several seaweed species of interest.

We envision using this reference culture collection in understanding the biology, life history, ecological adaptations, evolution, speciation, and biogeography of (tropical) seaweeds as well as to develop underutilized seaweeds in the country, among others.

Materials and Methods Sample collection

Abiotic natural substrata (e.g., old shells, rocks, coral fragments, pebbles), as well as portions of seagrass and seaweeds, were collected by snorkeling or SCUBA diving from the intertidal to subtidal areas of onshore and offshore reefs and features within the Philippine archipelago (Figures 1–3) from 2018 until 2022. Collecting tubes filled with sterile seawater or resealable plastic bags were used in sample collection. Samples were kept in tubes with sterile seawater with or without culture medium at room temperature and were brought to the laboratory for incubation, isolation, and maintenance in culture.

Establishment and maintenance of unialgal isolates

Collected natural substrata and crude cultures of seaweeds were placed either in 100 mm Petri dishes or in 500 mL glass vessels, both containing modified PES culture media (West & McBride 1999) and incubated according to the germling emergence method (GEM) outlined by Peters et al. (2015). Our efforts in establishing the seaweed reference culture collections were initiated in 2018, following the culture directions outlined by West (2005). Initially, f/2 culture medium (Guillard & Ryther 1962; Guillard 1975) was used. While our cultures grew well, we have had some serious issues with diatom and bacterial contamination. After which, we used Provasoli’s Enriched Natural Seawater medium (PES;

Provasoli 1968) but this would sometimes result in the stunted growth of red seaweeds. Since most of the red seaweeds in long-term in vitro culture are nitrogen-limited (West 2005), we adopted the modified PES medium developed by West

& McBride (1999) with some minor modifications such as increasing the amount of NaNO₃ to 4.0 g and removing boron from the PII trace metals mix. For maintaining crude cultures of green and brown seaweeds, autoclaved seawater (33–34 ppt) is enriched with 10 mL of the culture media; whereas for red seaweeds, 20 mL of modified PES medium per liter proved to encourage better growth. To minimize bacterial contamination, 30 mg each of penicillin G sodium salt (Sigma-

Aldrich, USA) and streptomycin sulfate (Sigma-Aldrich, USA) are added to the enriched seawater. Occasional growth of diatoms in crude cultures was dealt with GeO₂ (Sigma- Aldrich, USA) following the recommendation of West (2005).

All cultures are maintained under the following conditions: An ambient temperature of 23±2 ºC, under diffuse Daylight LED fluorescent tubes, where greens and browns are grown under 12.5–13 μmol photon m^-2s^-1 irradiance while reds are kept at 5 μmol photon m^-2s^-1, and a 12:12 light:dark photoperiod.

Emerging algae from the natural substrata are isolated by cutting or pipetting their fragments under a ZEISS Primovert Compact Inverted Microscope (Zeiss, Jena, Germany).

Changing culture media and cleaning of cultures are typically done every two weeks or, on some occasions, when culture vessels were visibly dirty. Where appropriate, samples are carefully cleaned using soft-bristled brushes under a stereomicroscope or inverted microscope.

Identification of isolates

Initial identification of each seaweed grown in vitro is primarily based on their morphological and anatomical characteristics. Where possible, these are identified at least at the genus level based on guides on tropical Indo-Pacific marine algae such as Trono (1997, 2004), Calumpong &

Meñez (1997), Coppejans et al. (2010, 2017), and Tsutsui et al.

(2005). These identifications are verified later by integrating morpho-anatomical observations with DNA barcodes.

DNA barcoding and molecular phylogenetic analyses of some isolates

Live cultures of some brown (Phaeophyceae) and red (Florideophyceae, particularly the family Delesseriaceae) seaweeds currently being maintained at UP-MMARCC were used for DNA barcoding and molecular phylogenetic analyses. Subsamples of tissue from live cultures were used for extraction of genomic DNA following the methods described in Santiañez et al. (2018). Polymerase chain reaction (PCR) was performed on the rbcL gene using Hot Start Taq 2X Master Mix (New England Biolabs, Inc., Massachusetts, USA). Primers and PCR conditions used in amplifying the rbcL gene markers for brown and red seaweeds are outlined in Table S1. PCR products were cleaned using Monarch® PCR &

DNA Cleanup Kit (New England Biolabs, Inc., Massachusetts, USA) and were sent to Macrogen, Inc. (Seoul, South Korea) for sequencing. Molecular phylogenetic trees based on newly generated and GenBank-available rbcL sequences (Table S2) were generated using Maximum Likelihood analyses following those described in Santiañez et al. (2018).

Results and Discussion

We are currently maintaining 446 seaweeds (Table 1) collected from 50 sampling stations in the Philippines (Figs.

1–4, Table 2). Of these, 265 are red (59%), 88 brown (20%), and 97 are green seaweeds (21%) (Figure 5). In addition, 25 unialgal Colpomenia sinuosa (Mertens ex Roth) Derbès

& Solier isolates from Okinawa, Japan (courtesy of Dr.

Masakazu Hoshino and Dr. Kazuhiro Kogame of Hokkaido University) are also being maintained in the laboratory.

Some common and/or interesting isolates of green, brown, and red seaweeds are also highlighted herein (Figs. 6–8). We

(3)

Philippine Journal of Systematic Biology Online ISSN: 2508-0342

Table 1. List of seaweeds currently being maintained at the UPMSI Marine Macroalgal Reference Culture Collection (UP-MMARCC).

Taxa¹ Number of samples

Ulvophyceae, Chlorophyta 93

Anadyomene 4

Boodlea 1

Boodleacean 4

Bryopsis 3

Caulerpa 3

Chaetomorpha 1

Cladophora 5

Cladophoracean 4

Codium 1

Dictyosphaeria 3

Halimeda 1

Lychaete 12

Rhipidosiphon 1

Struvea 10

Ulva 28

Ulva lactuca 1

Valonia 1

Valonia aegagropila 9

Valonia ventricosa 1

Phaeophyceae, Ochrophyta 88

Asterocladon 3

Brown epiphyte (unknown) 1

Colpomenia sp. 7

Colpomenia sinuosa ^JP 16

Colpomenia sinuosa 2 ^JP 4

Colpomenia sinuosa 3 ^JP 5

Dictyopteris 3

Dictyota 4

Ectocarpalean 1

Hydroclathrus 5

Padina 1

Pseudochnoospora 21

Spatoglossum 1

Sphacelaria 10 3

Sphacelaria 13 1

Sphacelaria 7 3

Sphacelaria rigidula 3

Sphacelaria tribuloides 3

Stypopodium 3

Florideophyceae, Rhodophyta 265

Acanthophora 2

Acrochaetiales 8

Amansia 16

Asparagopsis 2

Asparagopsis (Falkenbergia/Tetrasporophyte) 19

Asteromenia 1

Botryocladia 2

Callophycus 4

Ceramium 7

Ceratodictyon ('Gelidiopsis') 5

Chondrophycus 1

Crouania 2

Cryptonemia 6

Eucheuma denticulatum 3

Eucheuma denticulatum ('spinosum brown') 3

(4)

Gelidium 1

Gibsmithia 4

Gracilaria 14

Gracilaria ('flat') 1

Gracilaria salicornia 1

Griffithsia 13

Halymeniales 1

Hydropuntia eucheumatoides 1

Hypoglossum 1

Hypoglossum (335) 11

Hypoglossum (337) 6

Hypoglossum 1 4

Hypoglossum 2 4

Hypoglossum 3 4

Hypoglossum 4 4

Hypoglossum 5 4

Hypoglossum 6 4

Hypoglossum 7 4

Hypoglossum 8 4

Hypoglossum 9 4

Hypoglossum 10 4

Hypoglossum 11 4

Hypoglossum 12 4

Hypoglossum 13 1

Hypoglossum 14 1

Hypoglossum 15 1

Hypoglossum 16 1

Hypoglossum 17 1

Hypoglossum 18 1

Hypoglossum 19 1

Hypoglossum 20 1

Hypoglossum 21 1

Hypoglossum 22 1

Hypoglossum 23 1

Hypoglossum 24 1

Hypoglossum 25 1

Hypoglossum 26 1

Hypoglossum 27 1

Hypoglossum 28 1

Kappaphycus 5

Kappaphycus alvarezii (culture isolate) 7

Laurencia 4

Martensia 1

Melanothamnus 3

Neurymenia 7

Neurymenia (carpospores) 1

Nitophyllum 3

Peyssonnelid 2

Portieria 1

Red epiphyte (unknown) 8

Red seaweed (unknown) 3

Rhodomelacean 5

Solieriacean 3

Tolypiocladia 1

Vanvoorstia 4

Wrangelia 4

Yonagunia 1

Zellera tawallina 3

GRAND TOTAL 446

1 Names listed in this table are preliminary identifications based on gross morphology only.

JP Colpomenia sinuosa cultures from Japan.

(5)

Table 2. List of collection localities of seaweeds currently being maintained at the UP-MMARCC. Site numbers correspond to the marked stations in Figures 1–4.

Site no. Locality Municipality Province Number of cultures

1 Patar Bolinao Pangasinan 18

2 Estanza Bolinao Pangasinan 4

3 Balingasay Bolinao Pangasinan 3

4 Trenchera Bolinao Pangasinan 3

5 Arosan Bolinao Pangasinan 1

6 BML Beachfront Bolinao Pangasinan 8

7 Lucero Bolinao Pangasinan 20

8 Silaki Island Bolinao Pangasinan 7

9 Dos Hermanos Bolinao Pangasinan 5

10 MalWest Bolinao Pangasinan 1

11 Malilnep Bolinao Pangasinan 6

12 Goyoden Bolinao Pangasinan 3

13 Dewey Bolinao Pangasinan 20

14 Cangaluyan Anda Pangasinan 9

15 Magsaysay Anda Pangasinan 2

16 Almeida Balaoan La Union 4

17 Paraoir Balaoan La Union 2

18 Quezon Station 1 Quezon Palawan 9

19 Quezon Station 2 Quezon Palawan 6

20 Jackson Atoll Station 1 Kalayaan Palawan 3

21 Pagasa Island Station 4 Kalayaan Palawan 18

22 Pagasa Island Seagrass 4 Kalayaan Palawan 3

27 Ayungin Shoal Station 3 Kalayaan Palawan 12

28 Ayungin Shoal Station 2 Kalayaan Palawan 31

29 Sabina Shoal Station 1 Kalayaan Palawan 2

32 Aurora Station 1 Dilasag Aurora 2

33 Aurora Station 2 Dilasag Aurora 4

34 Casiguran Station 1 Casiguran Aurora 10

35 Casiguran Station 2 Casiguran Aurora 1

36 Aurora Station 4 Casiguran Aurora 5

37 Aurora Station 5 Casiguran Aurora 12

38 Aurora Station 8 Dipaculao Aurora 22

39 Aurora Station 9 Dipaculao Aurora 2

40 Aurora Station 13 Baler Aurora 4

41 Benham Bank, Philippine Rise 8

42 Casa Astillero Calatagan Batangas 15

43 Anilao Mabini Batangas 29

44 Talaga Mabini Batangas 2

45 Pagkilatan Batangas City Batangas 7

46 BSU–Lobo Lobo Batangas 2

47 Tabugon Santa Fe Romblon 3

48 USC Marine Station Maribago Cebu 9

Note: Donated culture samples such as Kappaphycus spp. and Colpomenia sinuosa isolates from Japan were not included in the above tally since they were not collected from the field.

were able to generate five sequences for brown seaweeds and another five for red seaweeds, particularly Delesseriaceae.

Our brown seaweed sequence data represented three brown algal families: the Scytosiphonaceae [Colpomenia (Endlicher) Derbès & Solier and Hydroclathrus Bory de Saint-Vincent], Asterocladaceae (a putative new species of Asterocladon D.G. Müller, E.R. Parodi & A.F. Peters), and Sphacelariaceae (Sphacelaria spp.) (Fig. 9). Among the delesseriacean algae

we have isolated from the West Philippine Sea area, we have found that four formed distinct lineages: two formed a clade with Zellera tawallina G. Martens, while the other three appear unique, each likely representing putative new species of Hypoglossum Kützing (Fig. 10).

Regarding green seaweeds, we highlighted here some common but understudied Philippine taxa such as Lychaete J. Agardh, Ulva lactuca Linnaeus, Valonia ventricosa J.

(6)

Figure 1. . Collection localities of specimens from Pangasinan, northwest Luzon that are currently housed at the UP-MMARCC.

Stations numbers correspond to those outlined in Table 1.

Figure 2. Collection localities of seaweeds from the Kalayaan Island Group and onshore stations in Quezon, Palawan that are being maintained at the UP-MMARCC. Stations numbers correspond to those outlined in Table 1.

(7)

Figure 3. Collection localities of seaweeds from the coasts of Aurora and the Philippine Rise that are deposited at the UP- MMARCC. Stations numbers correspond to those outlined in Table 1.

Figure 4. Other minor collection localities of seaweeds in the Philippines that are deposited at the UP-MMARCC. Stations numbers correspond to those outlined in Table 1.

(8)

Figure 5. The composition of seaweeds currently housed at the UP-MMARCC, classified according to the major groups and subdivided into orders.

Agardh, and Struvea Sonder (Fig. 6). A species of Struvea collected from 10 m deep on the reefs of Pag-asa Island (Site 25; Fig. 2)—a new species record for the island—is also being maintained in our cultures (Fig. 6E).

Our cultures of brown seaweeds include Dictyopteris J.V. Lamouroux, Spatoglossum Kützing, Sargassum C.

Agardh, Sphacelaria Lyngbye, and Colpomenia (Endlicher) Derbès & Solier (Fig. 7). Most of the taxa that we have isolated from the incubated substrata are minute, filamentous, brown seaweeds, which are among the most understudied seaweed groups in the Philippines. Of more than 1,000 seaweed taxa in the country, only 10 taxa of minute and filamentous brown seaweeds are reported (Lastimoso & Santiañez 2021). That is five for Acinetosporaceae, three species of Sphacelariaceae, and two for Ectocarpaceae. We attributed these low records to the lack of thorough taxonomic studies on the group. An integrative approach combining unialgal culture studies with molecular techniques (i.e., DNA barcoding) has shown to be successful in delineating species on these “cryptic” brown seaweeds such as Acinetospora spp. (Yaegashi et al. 2015), Ectocarpus spp. (Montecinos et al. 2017), and Sphacelaria spp. (Keum et al. 2005). More recently, an integrative study on Asterocladon spp. from Japan and the Philippines resulted in the first report of the genus as well as discovery of a new seaweed species named A. ednae Sasagawa, Santiañez, &

Kogame from Camotes Islands, Cebu, Philippines (Sasagawa et al. 2022). Regarding minute and filamentous brown seaweeds, we have focused our efforts on establishing unialgal cultures for our Sphacelaria samples mainly by selectively isolating

and growing unique propagules. Currently, four distinct Sphacelaria morphotypes are being maintained as unialgal cultures in UP-MMARCC. Of these, we obtained rbcL sequences from two isolates which allied with Sphacelaria tribuloides Meneghini (UPMMARCC-007) and Sphacelaria rigidula Kützing (UPMMARCC-001) (Fig. 9). Consequently, this verifies the occurrence of both species in the Philippines.

Based on their morphologies, the two other Sphacelaria species can be attributed to Sphacelaria novae-hollandiae Sonder (UPMMARCC-010), while the other one (UPMMARCC-004) remains unidentified. Aside from Sphacelaria, we also established unialgal cultures of a “cryptic”, filamentous, and epiphytic brown seaweed from C. sinuosa samples collected from Bolinao, Pangasinan. Based on rbcL sequence data, this isolate (UPMMARCC-118) is attributable to the genus Asterocladon and is most closely related to A. ednae. A more detailed taxonomic study is needed to ascertain whether this species should be attributed to A. ednae or described as a new and distinct species. In addition, we successfully isolated and currently maintain C. sinuosa (UPMMARCC-120) (Figs. 7E, 8) collected from Dewey, Bolinao, Pangasinan. Based on these isolates and other Colpomenia specimens collected from the northern West Philippine Sea region, we are currently looking into the molecular phylogeny, taxonomy, and life history in culture.

Among red seaweeds, we collected several representatives of relatively understudied taxa in the Philippines including Gibsmithia Doty, Hypoglossum Kützing, Amansia J.V. Lamouroux, Cryptonemia J. Agardh, Neurymenia J.

(9)

Figure 6. Green seaweed (Ulvophyceae, Chlorophyta) samples being maintained at UP-MMARCC. (A) Profusely branching Lychaete (UPMMARCC-0025) collected from Dewey, Bolinao, Pangasinan. (B) Foliose blade fragments of Ulva lactuca (UPMMARCC-0104) collected from the beachfront area of the UP-Bolinao Marine Laboratory, Bolinao, Pangasinan. (C) Cushion-forming siphonous Valonia aegagropila (UPMMARCC-0018) collected from the beachfront area of the UP-Bolinao Marine Laboratory, Bolinao, Pangasinan. (D) Dead coral fragment serving as substratum to several seaweeds, especially Valonia ventricosa (arrowhead) (UPMMARCC-0074) collected from Patar, Bolinao, Pangasinan. (E) Struvea collected from Sabina Shoal showing several stipitate blades. (F) Loosely branched Lychaete (UPMMARCC-0176) collected from Pag-asa Island, Kalayaan, Palawan. All scale bars = 1 cm.

(10)

Figure 7. Brown seaweed (Phaeophyceae, Ochrophyta) samples being maintained at UP-MMARCC. (A) Thin morphotype of Dictyota (UPMMARCC-0145) collected in the northeastern seagrass area of Pag-asa Island, Kalayaan, Palawan. Scale bar = 140 μm. (B) Dictyopteris samples collected from ‘MalWest’, Bolinao, Pangasinan. Scale bar = 140 μm. (C) Uniseriate filaments of the epiphytic Asterocladon sp.

(UPMMARCC-0118) collected from Dewey, Bolinao, Pangasinan with a stellate-arrangement of chloroplast around its prominent pyrenoid (inset). Scale bar = 35 μm, scale bar = 14 μm (inset). (D) Sphacelaria tribuloides (UPMMARCC-0007) propagule isolated from a crude culture collected from Goyoden, Bolinao. Scale bar = 50 μm. (E) Saccate thallus of a Colpomenia sp. (UPMMARCC-0120) collected from Dewey, Bolinao, Pangasinan. Scale bar = 140 μm. (F) Portion of blade fragments of strap-shaped and branching Spatoglossum (UPMMARCC-0114) collected from Dewey, Bolinao, Pangasinan. Scale bar = 1 cm.

Agardh, Botryocladia (J. Agardh) Kylin, Asparagopsis Montagne (including its tetrasporic or “Falkenbergia”

stage), and Laurencia J.V. Lamouroux (Fig. 8). Of these, we are currently focusing our work on the two Hypoglossum samples (UPMMARCC-214 and 215) collected from Sabina Shoal in 2019 that show two distinct life history stages. One isolate (UPMMARCC-0215) is a female gametophytic plant (identified through the presence of cystocarps) that profusely grows through fragmentation (Fig. 8D). The other one is a diploid tetrasporic plant (UPMMARCC-0214) that grows

slower than the female gametophyte. This tetrasporic plant released tetraspores that eventually settled at the bottom of the culture dishes. From these haploid tetraspores, we isolated and raised male and female gametophytes. These new generations of gametophytic thalli have since matured as seen in their production of reproductive structures (Figs. 8E–F).

To further delineate the Hypoglossum species found in the Indo-Pacific, three new additional Hypoglossum morphotypes (UPMMARCC-240, 243, and 244) were collected during the 2021 West Philippine Sea research expedition and are

(11)

Philippine Journal of Systematic Biology Online ISSN: 2508-0342 Figure 8. Selected red seaweed

(Florideophyceae, Rhodophyta) samples being maintained at UP-MMARCC. (A) Branching and ‘hairy’ Gibsmithia (UPMMARCC-0069) collected from Magsaysay, Anda, Pangasinan. Scale bar = 1 cm. (B) Portion of a branch of Amansia (UPMMARCC-0165) collected from Station 1 in Quezon, Palawan. Scale bar = 1 cm. (C) Foliose and branched Cryptonemia (UPMMARCC-0164) growing on a pebble collected from Pag-asa Island, Kalayaan, Palawan. Scale bar = 1 cm. (D) Gametophytic Hypoglossum (UPMMARCC-215) collected from Sabina Shoal. Scale bar

= 1 cm. (E) Carpospores being released from the cystocarp of a female Hypoglossum (UPMMARCC-214) collected from Sabina Shoal. Scale bar = 140 μm. (F) Portion of a branch of the male gametophyte of Hypoglossum (UPMMARCC-214) showing spermatangial sori. Scale bar = 140 μm. (G) Flattened Gracilaria sp (UPMMARCC-0083).

collected from Lucero, Bolinao, Pangasinan. Scale bar = 1 cm. (H) Old portion of a branch of a Neurymenia ( U P M M A R C C - 0 0 5 1 ) collected from Cangaluyan, Anda, Pangasinan showing new branchlets. Scale bar = 1 cm. (I) Pyriform vesicles of several Botryocladia (UPMMARCC-0082) branchlets collected from Patar, Bolinao, Pangasinan. Scale bar = 1 cm. (J) Tetrasporophyte (Falkenbergia stage) of the economically important Asparagopsis taxiformis (UPMMARCC-0066) collected from Patar, Bolinao, Pangasinan. Scale bar = 1 cm.

(K) Branching Laurencia (UPMMARCC-0057) isolated from a Dictyosphaeria sample collected from Almeida, Balaoan, La Union. Scale bar = 1 cm.

(12)

currently being maintained and examined in the laboratory.

GEM (Peters et al. 2015) also proved to be effective in allowing the growth of some emergent red seaweeds. In 2019, a sample of the green seaweed Dictyosphaeria was collected from the intertidal of Almeida, Balaoan, La Union (Station 16; Fig. 1). It was kept under in vitro conditions for almost a year and in 2020, we noticed an epiphytic red seaweed emerging in between the bubble-shaped cells of the thallus of the Dictyosphaeria. The epiphyte was isolated on 20 February 2020 and maintained in the laboratory. After a year, the Dictyopshaeria specimen expired (because of the intermittent

maintenance due to the ongoing pandemic); however, the epiphytic red seaweed, now tentatively identified as a species of Laurencia (UPMMARCC-0057) (Fig. 8K), continued to proliferate, liberated spores during the first quarter of 2021, and now has four unialgal isolates.

Strains and isolates at the UP-MMARCC can be freely accessed and obtained only for scientific and non-commercial purposes, subject to existing pertinent national and international laws. To facilitate the growth of our collection, we also welcome the exchange and deposits of seaweed strains in the Philippines and other tropical areas. For details

Figure 9. Maximum likelihood (ML) molecular phylogenetic tree of some members of the Phaeophyceae based on rbcL sequences including some taxa of culture isolates being maintained in UP-MMARCC (indicated in bold text). ML bootstrap values <50% were removed.

(13)

Figure 10. Maximum likelihood (ML) molecular phylogenetic tree of the family Delesseriaceae based on rbcL sequences including some taxa of culture isolates being maintained in UP-MMARCC (indicated in bold text). ML bootstrap values <50% were removed.

on specimens’ information: codes, strain numbers, collection dates, localities (GPS coordinates), etc., as well as, accessing, obtaining, exchanging, and/or depositing strains and isolates at UP-MMARCC, we encourage interested parties to directly contact the senior author.

We showed herein the potential of UP-MMARCC in uncovering Philippine seaweed biodiversity resources and its capacity to safeguard not only the unutilized and underdeveloped Philippine seaweed species (e.g., Ulva spp., A. taxiformis, Sargassum spp.) but also possible novel strains and haplotypes of economically important species in the country (e.g., Kappaphycus spp, and Gracilaria spp.). As such, we anticipate tapping and maximizing the potentials of

the UP-MMARCC as a biobank for economically important Philippine seaweeds as part of our continued efforts to support the development of the Philippine seaweed industry. Towards that end, our recent work is focused on understanding the in vitro and land-based nursery culture requirements of A.

taxiformis and Ulva species based on the isolates deposited at UP-MMARCC.

Presently, the contents of UP-MMARCC are primarily from the West Philippine Sea area, and we are planning to broaden the geographical scope of our sampling areas to cover other marine biogeographic regions in the Philippines.

Nonetheless, the UP-MMARCC, to our knowledge, is the most widely sampled and holds the most diverse collection

(14)

of live seaweeds under in vitro culture in the Philippines so far. As such, our culture collection opens possibilities to advance phycological research in the Philippines, especially in understanding the biology and physiology of seaweeds as well as in elucidating and developing their derivatives for various applications.

Conclusion

The UPMSI Marine Macroalgal Reference Culture Collection (UP-MMARCC), to the best of our knowledge, holds the most diverse and widely sampled collection of living seaweeds being maintained in vitro in the Philippines.

As we have outlined here, the UP-MMARCC is facilitating documentation of often overlooked components of the Philippine seaweed flora, including helping with the discovery of new and/or unreported taxa. Considering the possibilities for phycological research being offered by our culture collection, we anticipate that in the coming years, it will play an important role in understanding several aspects of tropical seaweed biology, especially their life histories and reproduction. The crucial role of the UP-MMARCC is also underscored by its contribution to the understanding of the ecologic importance and economic potentials of the resources of key marine biodiversity areas and marine biogeographic regions in the country, such as the West Philippine Sea.

This is particularly true for the relatively underexplored and understudied Kalayaan Island Group. As such, we hope that with continued funding support, we can maintain and sustain these living resources in our reference culture collection not only for research but especially in support of the seaweed industry and in diversifying their products.

Acknowledgments

We thank Dr. Edna T. Ganzon-Fortes and Dr. Gavino C. Trono Jr. for their dedicated work on the biodiversity and culture of Philippine seaweeds that inspired this current effort to establish the UP-MMARCC. WJES acknowledges the Office of the Chancellor of the University of the Philippines Diliman, through the Office of the Vice-Chancellor for Research and Development, for funding support in establishing and maintaining UP-MMARCC through the Ph.D. Incentive Award (Project Nos. 191926 PhDIA and 202104 PhDIA Y2) and the Balik Scientist Program of the Philippine Department of Science and Technology. JMLL is grateful for the LinnéSys:

Systematic Research Fund from the Systematics Association and The Linnean Society of London for funding the preliminary molecular work on the delesseriacean culture isolates. We also thank the UPMSI in-house research grant for the additional funding support in maintaining the UP-MMARCC and the UPMSI and UPMSI-Bolinao Marine Laboratory staff for their assistance in securing the seawater we use in our culture during the pandemic. We are indebted to Dr. Masakazu Hoshino and Dr. Kazuhiro Kogame (Hokkaido University, Japan) and Dr. John A. West (University of Melbourne, Australia) for sending their scytosiphonacean samples to the Philippines and Dr. Alvin P. Monotilla (University of San Carlos, Cebu) for generously sharing specimens collected along the coast of the USC Marine Station. Collection trips in the offshore island

reefs of the Kalayaan Island Group, as well as in the onshore areas of Palawan, the Philippine Rise, and along the coasts of Aurora were funded by the National Security Council through the Upgrading Capacity, Infrastructure, and Assets for Marine Science Research in the Philippines (UPGRADE CIA) project and by the Biodiversity Management Bureau of the Department of Environment and Natural Resources through the Predicting Responses between Ocean Transport and Ecological Connectivity of Threatened ecosystems in the West Philippines Sea (PROTECT WPS 1 & 2) (2019–2020) and the Surveying and Assessing Valuable Ecosystems and seascapes in Philippine Rise (SAVE PhilRise) (2021–2022) projects. Lastly, sample collection would not have been possible without the daily research support of the ship crew of RV Panata and the dive assistance of Rhea Mae A. Luciano, Christine S. Segumalian, Jeremiah Noelle C. Requilme, Lala Grace E. Calle, Dana P. Manogan, and Dr. Hazel O. Arceo for the West Philippine Sea collections; Steffi Mari J. Arceta for the Aurora collections; and Mary Joyce P. Velos along with the PA0522 researchers for our Benham Bank samples. We are greatly indebted to Dr. Rachel Ravago-Gotanco and her team (the members of the Marine Molecular Ecology and Evolution Laboratory) at the UPMSI for generously sharing their molecular laboratory and its facilities allowing us to generate the necessary genetic information in this study.

Literature Cited

Aaron-Amper J., D.B. Largo, E.R.B. Handugan, J.I. Nini, K.M.S. Alingasa, & S.J. Gulayan, 2020. Culture of the tropical brown seaweed Sargassum aquifolium: From hatchery to field out-planting. Aquaculture Reports, 16:

100265.

Abe T., A. Kurihara, S. Kawaguchi, R. Terada, & M. Masuda, 2006. Preliminary report on the molecular phylogeny of the Laurencia complex (Rhodomelaceae). Coastal Marine Science, 30: 209–213.

Bittner L., C. Payri, A. Couloux, C. Cruaud, B. Dereviers, & F.

Rousseau, 2008. Molecular phylogeny of the Dictyotales and their position within the Phaeophyceae, based on nuclear, plastid and mitochondrial DNA sequence data.

Molecular Phylogenetics and Evolution, 49(1): 211–226.

Calumpong H.P. & E.G. Meñez, 1997. Field Guide to the Common Mangroves, Seagrasses and Algae of the Philippines. Bookmark, Philippines. 197 pp.

Campbell C.N. & M. Lorenz, 2020. Algal culture collections in the -omics age. Applied Phycology, 1(SI1): 1–2.

Coppejans E., A. Prathep, F. Leliaert, K. Lewmanomont, &

O. De Clerck, 2010. Seaweeds of Mu Ko Tha Lae Tai (SE Thailand): Methodologies and Field Guide to the Dominant Species. Biodiversity Research and Training Program, Bangkok, Thailand. 274 pp.

Coppejans E., A. Prathep, K. Lewmanomont, K. Hayashizaki, O. De Clerck, F. Leliaert, & R. Terada, 2017. Seaweeds and Seagrasses of the Southern Andaman Sea Coast of Thailand. Kagoshima University Museum, Kagoshima, Japan. 244 pp.

Dawes C.J. & E.W. Koch, 1991. Branch, micropropagule and tissue culture of the red algae Eucheuma denticulatum and Kappaphycus alvarezii farmed in the Philippines.

Journal of Applied Phycology, 3: 247–257.

(15)

Philippine Journal of Systematic Biology Online ISSN: 2508-0342 Dawes C.J., A.O. Lluisma, & G.C. Trono Jr., 1994. Laboratory

and field growth studies of commercial strains of Eucheuma denticulatum and Kappaphycus alvarezii in the Philippines. Journal of Applied Phycology, 6: 21–24.

Dawes C.J., G.C. Trono Jr., & A.O. Lluisma, 1993. Clonal propagation of Eucheuma denticulatum and Kappaphycus alvarezii for Philippine seaweed farms. Hydrobiologia, 260: 379–383.

Draisma S.G.A., W.F. Prud’Homme van Reine, W.T. Stam,

& J.L. Olsen, 2001. A reassessment of phylogenetic relationships within the Phaeophyceae based on RUBISCO large subunit and ribosomal DNA sequences.

Journal of Phycology, 37(4): 586–603.

Freshwater D.W. & J. Rueness, 1994. Phylogenetic relationships of some European Gelidium (Gelidiales, Rhodophyta) species, based on rbcL nucleotide sequence analysis.

Phycologia, 33(3): 187–194.

Ganzon-Fortes E.T., 2012. A historical account of biodiversity studies on Philippine seaweeds (1800–1999). Coastal Marine Science, 35(1): 182–201.

Ganzon-Fortes E.T. & W.J.E. Santiañez, 2021. The Gregorio T. Velasquez Phycological Herbarium (MSI): A Legacy and National Heritage. Philippine Journal of Science, 151(S1): 1–7.

Guillard R.R.L., 1975. Culture of phytoplankton for feeding marine invertebrates. In: Smith W.L. & Chanley M.H., (eds.), Culture of Marine Invertebrate Animals. Springer, New York. 29–60.

Guillard R.R.L. & J.H. Ryther, 1962. Studies of marine planktonic diatoms: I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran. Canadian Journal of Microbiology, 8(2): 229–239.

Kawai H., T. Hanyuda, S. Akita, & S. Uwai, 2020. The macroalgal culture collection in Kobe University (KU- MACC) and a comprehensive molecular phylogeny of macroalgae based on the culture strains. Applied Phycology, 1(SI1): 1–8.

Keum Y.-S., J.-H. Oak, S.G.A. Draisma, W.F. Prud’homme van Reine, & I.-K. Lee, 2005. Taxonomic reappraisal of Sphacelaria rigidula and S. fusca (Sphacelariales, Phaeophyceae) based on morphology and molecular data with special reference to S. didichotoma. Algae, 20(1):

1–13.

Kogame K., T. Horiguchi, & M. Masuda, 1999. Phylogeny of the order Scytosiphonales (Phaeophyceae) based on DNA sequences of rbcL, partial rbcS, and partial LSU nrDNA. Phycologia, 38(6): 496–502.

Lastimoso J.M.L. & Santiañez W.J.E., 2021. Updated checklist of the benthic marine macroalgae of the Philippines.

Philippine Journal of Science, 150(S1): 29–92.

Luhan M.R.J. & H. Sollesta, 2010. Growing the reproductive cells (carpospores) of the seaweed, Kappaphycus striatum, in the laboratory until outplanting in the field and maturation to tetrasporophyte. Journal of Applied Phycology, 22: 579–585.

Montecinos A.E., L. Couceiro, A.F. Peters, A. Desrut, M.

Valero, & M.-L. Guillemin, 2017. Species delimitation and phylogeographic analyses in the Ectocarpus subgroup siliculosi (Ectocarpales, Phaeophyceae).

Journal of Phycology, 53(1): 17–31.

Nelson W., J. Dalen, & K. Neill, 2013. Insights from natural

history collections: analysing the New Zealand macroalgal flora using herbarium data. Phytokeys, 30:

1–21.

Peters A.F., L. Couceiro, K. Tsiamis, F.C. Küpper, & M.

Valero, 2015. Barcoding of cryptic stages of marine brown algae isolated from incubated substratum reveals high diversity in Acinetosporaceae (Ectocarpales, Phaeophyceae). Cryptogamie, Algologie, 36(1): 3–29.

Provasoli L., 1968. Media and prospects for the cultivation of marine algae. In: Watanabe A. & A. Hattori, (eds.), Cultures and Collections of Algae. Proceedings of the U.S.–Japan Conference. Japanese Society of Plant Physiology, Hakone, Japan. 63–75.

Rula N.A.M., E.T. Ganzon-Fortes, M.J.R. Pante, & G.C. Trono Jr., 2021. Influence of light, water motion, and stocking density on the growth and pigment content of Halymenia durvillei (Rhodophyceae) under laboratory conditions.

Journal of Applied Phycology, 33(4): 2367–2377.

Santiañez, W.J.E., K.M. Lee, S. Uwai, A. Kurihara, P.J.L. Geraldino, E.T. Ganzon-Fortes, S.M. Boo, &

K. Kogame, 2018. Untangling nets: Elucidating the diversity and phylogeny of the clathrate brown algal genus Hydroclathrus, with the description of a new genus Tronoella (Scytosiphonaceae, Phaeophyceae).

Phycologia, 57(1): 61–78.

Santiañez W.J.E., H.J. Suan-Flandez, & G.C. Trono Jr., 2016.

White rot disease and epiphytism on Halymenia durvillei Bory de Saint-Vincent (Halymeniaceae, Rhodophyta) in culture. Science Diliman, 28(1): 54–60.

Sasagawa E., W.J.E. Santiañez, & K. Kogame, 2022.

Asterocladon ednae sp. nov. (Asterocladales, Phaeophyceae) from the Philippines. Phycological Research, (online copy): 1–7.

Starr R.C. & J.A. Zeikus, 1993. UTEX-The culture collection of algae at the University of Texas at Austin. Journal of Phycology, 29: 1–106.

Sutti S., M. Tani, Y. Yamagishi, T. Abe, K.A. Miller, & K.

Kogame, 2018. Neochondria gen. nov. (Rhodomelaceae, Rhodophyta), a segregate of Chondria, including N.

ammophila sp. nov. and N. nidifica comb. nov. Phycologia, 57(3): 262–272

Trono G.C. Jr., 1997. Field Guide and Atlas of the Seaweed Resources of the Philippines. Bookmark, Philippines.

306 pp.

Trono G.C. Jr., 2004. Field Guide and Atlas of the Seaweed Resources of the Philippines: Volume 2. DA-BAR:

Marine Environmental Resources Foundation, Philippines. 261 pp.

Trono G.C. Jr., 2014. Mariculture of the Red Alga Halymenia durvillaei Bory De Saint Vincent: Techniques from Spores to Sea Outplanting. Bureau of Agricultural Research, Department of Agriculture, Philippines. 10 pp.

Tsutsui I., Q.N. Huybh, H.D. Nguyen, S. Arai, & T. Yoshida, 2005. The Common Marine Plants of Southern Vietnam.

Japan Seaweed Association, Usa, Japan. 250 pp.

West J.A., 2005. Long-term macroalgal culture maintenance.

In: Andersen, R.A., (ed.), Algal Culturing Techniques.

Elsevier, USA. 157–163.

West J.A. & D. L. McBride, 1999. Long-term and diurnal carpospore discharge patterns in the Ceramiaceae, Rhodomelaceae and Delesseriaceae (Rhodophyta).

(16)

32 | © Assocation of Systematic Biologists in the Philippines Volume 16 Issue 1 - 2022 Hydrobiologia, 398/399: 101–113.

Yaegashi K., Y. Yamagishi, S. Uwai, T. Abe, W.J.E.

Santiañez, & K. Kogame, 2015. Two species of the genus Acinetospora (Ectocarpales, Phaeophyceae) from Japan: A. filamentosa comb. nov. and A. asiatica sp. nov.

Botanica Marina, 58(5): 331–343.

Supplementary Information

Tables S1–S2 are available from the corresponding author upon request.