Gall Structure and Specificity in Bostrychia Culture Isolates (Rhodomelaceae, Rhodophyta)

Home , Bostrychia (alga), Woolooware Bay

Research Article

Algae 2013, 28(1): 83-92 http://dx.doi.org/10.4490/algae.2013.28.1.083 Open Access

Gall structure and specificity in Bostrychia culture isolates (Rhodomelaceae, Rhodophyta)

John A. West1,*, Curt M. Pueschel2, Tatyana A. Klochkova3, Gwang Hoon Kim3, Susan de Goër4 and Giuseppe C. Zuccarello5 1School of Botany, University of Melbourne, Parkville VIC 3010, Australia 2Department of Biological Sciences, State University of New York at Binghamton, Binghamton, NY 13902-6000, USA 3Department of Biology, Kongju National University, Kongju 314-701, Korea 411 Rue des Moguerou, 29680 Roscoff, France 5School of Biological Sciences, Victoria University of Wellington, PO Box 600, Wellington, 6140, New Zealand

The descriptions of galls, or tumors, in red algae have been sparse. Kützing (1865) observed possible galls of Bostrychia but only presented a drawing. Intensive culture observations of hundreds of specimens of the genus Bostrychia over many years have revealed that galls appeared in only a small subset of our unialgal cultures of B. kelanensis, Bostrychia moritziana / radicans, B. radicosa, B. simpliciuscula, and B. tenella and continued to be produced intermittently or continuously over many years in some cultures but were never seen in field specimens. Galls appeared as unorganized tissue found primarily on males and bisexuals, but occasionally on females and tetrasporophytes. The gall cells usually were less pigmented than neighboring tissue, but contained cells with fluorescent plastids and nuclei. The galls were not trans- ferable to other potential hosts. Galls could be produced from gall-free tissue of cultures that originally had galls even after transfer to new culture dishes. Electon microscopy of galls on one isolate (3895) showed that virus-like particles are observed in some gall cells. It is possible that a virus is the causative agent of these galls.

Key Words: Bostrychia; galls; Rhodomelaceae; Rhodophyta; unialgal culture; virus-like particles

Introduction

Galls, or tumors, are unorganized tissue on otherwise (Bory de Saint-Vincent) Fredericq (as Iridaea laminarioi- normal plants. Galls are usually associated with abnor- des Bory de Saint-Vincent) (Correa et al. 1993). Bacteria mal cell division patterns and / or cell enlargement (Apt were seen in the galls of the red algae Chondracanthus 1988, Scheffer 1997). Galls have not been reported ex- teedei (Mertens ex Roth) Kützing (as Gigartina teedii tensively in red algae possibly because they are rare and [Roth] Lamouroux) (Tsekos 1982), Grateloupia ameri- not considered important, but the deformation of tissue cana Kawaguchi et Wang (as Prionitis lanceolata [Harvey] could have fitness consequences for the host. While the Harvey), and Polyneuropsis stolonifera M. J. Wynne, D. L. cause of all red algal galls is not known several causative McBride & J. A. West (McBride et al. 1974). Subsequently agents have been shown or suggested. Cyanobacteria bacteria were proven to be the cause of galls on G. ameri- were reported to cause galls in Mazzaella laminarioides cana (Apt and Gibor 1989, Ashen and Goff 1996, 1998,

This is an Open Access article distributed under the terms of the Received November 19, 2012, Accepted February 1, 2013 Creative Commons Attribution Non-Commercial License (http://cre- Corresponding Author ativecommons.org/licenses/by-nc/3.0/) which permits unrestricted * non-commercial use, distribution, and reproduction in any medium, E-mail: [email protected] provided the original work is properly cited. Tel: +61-3-8344-8080

2000, Ashen et al. 1999). A fungus possibly causes galls of rochrome DAPI (4′,6-diamidino-2-phenylindole, Sigma- Catenella nipae Zanardini (Zuccarello 2008). The galls of Aldrich, St. Louis, MO, USA) using a heat fixation method. Prionitis and Catenella were originally described as red Algal thalli were dipped in 5 µg mL-1 DAPI solution in algal parasites (Lobocolax deformans Howe and Catenel- seawater for 5 min, and then the cover slips were slightly locolax leeuwenii Weber-van Bosse, respectively). Experi- heated over a boiler for a few seconds. After staining, algal mental infection and formation of galls was not achieved thalli were mounted on slides in the DAPI solution and with fungi in Catenella nipae (Zuccarello 2008). were examined under a UV filter. Galls have also been associated with viruses. Virus- Micrographs were taken with Olympus DP50 digital like particles (VLPs) were found in the galls of Gracilaria camera affixed to an Olympus BX50 microscope (Olym- epihippisora Hoyle (Apt and Gibor 1991). In this species pus, Tokyo, Japan) using Viewfinder Lite and Studio Lite gall tissue was capable of autonomous growth but only in computer programs (Better Light Inc., Placerville, CA, an undifferentiated state. Pueschel (1995) also observed USA) or with a Zeiss GFL bright field microscope (Carl VLPs in the filamentous red alga Acrochaetium savianum Zeiss AG, Oberkochen, Germany) using a Canon G3 cam- (Meneghini) Nägeli (as Audouinella saviana). era (Canon Inc., Tokyo, Japan) and Photoshop CS4 com- We have studied Bostrychia over many years and it has puter program ( http://www.adobe.com/au/). become useful in research on speciation, ecophysiology, evolution and cell biology / video microscopy of repro- Transmission electron microscopy (TEM) duction (reviewed in Zuccarello and West 2011). A very large collection of over 1,000 isolates of all the recognized Galls were fixed in phosphate buffered saline (PBS) buf- species of Bostrychia has been established (http://www. fer containing 2% glutaraldehyde at 4°C for 2 h. The glu- botany.unimelb.edu.au/West). A small subset of these taraldehyde was then rinsed out with PBS buffer and the isolates has produced undifferentiated tissue that persist- cells were postfixed with %2 osmium tetroxide at 4°C for ed in culture. Galls of Bostrychia were observed in 1865, 1.5 h. Thereafter, the cells were rinsed out with PBS buffer when Kützing illustrated many species of Bostrychia and and were dehydrated in a graded acetone series, embed- depicted a gall on B. cornifera Montagne (Kützing 1865, ded in Spurr’s epoxy resin (Spurr 1969) and polymerized Pl. 24) (Fig. 1A in this paper), later synonymized with B. overnight in a 70°C oven (Polysciences Inc., Warrington, moritziana (Sonders ex Kützing) J. Agardh (King and Put- PA, USA). Sections stained with uranyl acetate and Reyn- tock 1989). Our observations on Bostrychia galls are pre- olds’s lead citrate (Reynolds 1963) were viewed and pho- sented below. tographed on a Phillips Bio Twin Transmission Electron Microscope (Phillips Electron Optics, Eindhoven, Nether- lands). We were able to carry out TEM studies on only one MATERIALS AND METHODS B. simpliciuscula isolate (3895).

Algal material and laboratory culture RESULTS Unialgal culture methods were described in West and Zuccarello (1999) and West (2005). Culture isolates were Refer to Table 1 for the time periods (years) in which all maintained at 18-23°C, 12 : 12 LD daily photoperiods, galls were seen in the various isolates. These galls were 3-5 µmol photons m-2 s-1 cool white fluorescent or LED predominantly found in isolates from Australia where lighting, MPM/2 culture medium (30‰ salinity). For fast- most isolates were obtained. Only a small percentage of er growth and reproduction cultures were placed in 10-15 our isolates had galls and they were only observed in B. µmol photons m-2 s-1 on rotary shaker (70 rpm). simpliciuscula Harvey ex J. Agardh, B. moritziana / radi- Most isolates used for this research program are now cans complex, B. radicosa (Itono) J. A. West, G. C. Zucca- available at the Korean Marine Plants Collection, Chun- rello & M. H. Hommersand, B. kelanensis Grunow, and B. gnam National University, 220 Gung-dong, Yuseong-gu, tenella (Lamouroux) J. Agardh. Daejeon, Korea. Bostrychia simpliciuscula Bright field and fluorescence microscopy B. simpliciuscula is a polyphyletic species consisting Algal nuclei were stained with the DNA-specific fluo- of three lineages (Zuccarello et al. 1999, Zuccarello and

http://dx.doi.org/10.4490/algae.2013.28.1.083 84 West et al. Galls of Bostrychia

A B C

D E F

G H

Fig. 1. Morphology of galls in Bostrychia. (A) Drawing of B. cornifera (currently B. moritziana) with several ‘galls’ developing on a monosiphonous branch (arrows). Reproduced from Kützing (1865). (B-E) B. simpliciuscula microscopic images of galls developing on polysiphonous axis and laterals in different isolates (3895, 3931, and 3910). (D & E) Through-focus images of the same gall (3910). (F) B. simpliciuscula, fluorescent DAPI staining of the gall cells nuclei (blue color, DAPI-stained nuclei; red color, plastid autofluorescence; arrow points to the dead cells inside the gall). (G & H) B. simpliciuscula, transmission electron microscopy images of virus-like particles in gall tissues of isolate 3895. Scale bars represent: B-F, 50 µm; G & H, 200 nm.

85 http://e-algae.kr Algae 2013, 28(1): 83-92

Table 1. Bostrychia moritziana / radicans, B. kelanensis, B. radicosa, B. simpliciuscula, and B. tenella isolates observed. Rubisco spacer lineages of B. moritziana / radicans and B. simpliciuscula from Zuccarello and West (2003, 2006), Zuccarello et al. (1999) or during this study Species Culture Collection site / Date Phases in Galls Lineage / No. culture Haplotype B. moritziana / 2746 Tooradin, Western Port Bay, VIC, AUS; Oct 25, 1986 T M F No 1/AU radicans 2748 Tooradin, Western Port Bay, VIC, AUS; Oct 25, 1986 T M F M 1999 1/AU 2749 Tooradin, Western Port Bay, VIC, AUS; Oct 25, 1986 T M F M 1989 1/AU 3204 Beachwood, Natal, ZFA; Oct 4, 1991 T M F F 1997-2012 1/AU 3571 Stuarts Point, NSW, AUS; Oct 2, 1995 T M F B M 2000-2005 1/AU 3668 Clyde River, Nelligen, NSW, AUS; Dec 13, 1996 T M F B M B 2000-2008 1/AU 3669 Clyde River, Nelligen, NSW, AUS; Dec 13, 1996 T M F M 1998-2004 1/AU 3673 Clyde River, Nelligen, NSW, AUS; Dec 13, 1996 T M F B MBF 1998-2010 1/AU 3675 Rhyll, Phillip Island, VIC, AUS; Dec 27, 1996 T M F M 2000-2003 1/AU 3676 Rhyll, Phillip Island, VIC, AUS; Dec 27, 1996 T M F M 1998-2012 1/AU 3679 Mooney Mooney, Hawkesbury River, NSW, AUS; Feb 11, 1997 T M F No 1/AU 3680 Thorsby Creek, Newcastle, NSW, AUS; Feb 11, 1997 T M F M 2002, 2006 1/AU 3813 Snowy River estuary, Malo, VIC, AUS; Dec 29, 1997 T M F M 2002, 2006 No data 3879 Bridge crossing, Calliope R, Gladstone, QLD, AUS; Jan 25, 1998 T M F M 2002-2005 No data 3905 Foster Beach, VIC, AUS; Dec 7, 1998 T M F M 2002 No data 3913 Port Albert Wharf, N. Island, NZL; Dec 16, 1998 T M F M 2002-2008 No data 3914 Helensville R. Reserve, N. Island, NZL; Dec 16, 1998 T M F M 2001-2002 3941 On Avicennia, Cowleds Landing, SA, AUS; Feb 10, 1999 M B M 2000, 1/AU B 2001, 2002 3968 On Sonneratia, Nusa Lembongan, IDN; Apr 25, 1999 T M F M 2000-2006, 1/AU 2012 3026 Bahia Magdalena, Baja California Sur, MEX; Jan 7, 1990 T M F F 1995, 2000, 5/A1 2002 3492 Jetty at Port Aransas, TX, USA, coll. B. Baca; Sep 15, 1974 T M F T 2002 5/B 3973 Nusa Lembongan, IDN; Apr 25, 1999 M B B 2002 6/NA 3998 Tomkinson River, Arnhem Land, NT, AUS; Aug 23, 1999 T M F B T 2002 No data 4124 Cedar Key, FL, USA; Sep 19, 2000 T M F F 2001-2009, 6/D M T 2002 B. kelanensis 3810 Cossack, WA, AUS, Dec 9, 1997 T M F M 2012 No data B. radicosa 4086 Tempusak (near Kota Belud), Sabah, MYS; Aug 13, 2000 T M F B B 2002 B. radicosa haplotype 4614 Okat Harbor, Kosrae, FSM; Feb 8, 2006 veg No B. radicosa haplotype 4621 Pacific Tree Lodge, Kosrae, FSM; Feb 8, 2006 veg No B. radicosa haplotype 4627 Fefan I. Chuuk, FSM; Feb 10, 2006 veg No B. radicosa haplotype 4663 Peniyak Village Weno I. Chuuk, FSM; Feb 11, 2006 veg No B. radicosa haplotype B. simplicius- 2747 Tooradin,Western Port Bay, VIC, AUS; Oct 25, 1986 T No H1 cula 2853 Port Welshpool, VIC, AUS; Dec 22, 1987 T No H1 2914 Torrens Island, SA, AUS; Sep 22, 1988 T No H1 2915 Torrens Island, SA, AUS; Sep 22, 1988 T No H1 2933 Millers Landing, Wilsons Promontory, VIC, AUS; Dec 17, 1988 T No H1 3108 Williamstown, VIC, AUS; Dec 30, 1990 T M F No H1 3109 Sydney, New South Wales NSW, AUS; Jan 4, 1991 M 1999, 2002 H2 3110 Sydney, NSW, AUS; Jan 4, 1991 T M F B M 2003 H2 3111 Sydney, NSW, AUS; Jan 4, 1991 F 1995, 1998 H2 3114 Sydney, NSW, AUS; Jan 4, 1991 T M F B M 1998-1999, H2 2001-2003 3298 Moruya R., NSW, AUS; Jan 11, 1993 T No H1 3304 Merimbula, NSW, AUS; Jan 12, 1993 T M F B No No data 3305 Bermagui R., NSW, AUS; Jan 12, 1993 T No H1 3306 Batemans Bay, NSW, AUS; Jan 12, 1993 T No H1

http://dx.doi.org/10.4490/algae.2013.28.1.083 86 West et al. Galls of Bostrychia

Table 1. continued

Species Culture Collection site / Date Phases in Galls Lineage / No. culture Haplotype 3308 Broughton Ck., NSW, AUS; Jan 11, 1993 T M F T 1995, M 1998- H2 1999, 2002, F 1996, 1998, 2002 3315 Tuross Lake, NSW, AUS; Jan 12, 1993 T No H1 3317 Narooma, NSW, AUS; Jan 12, 1993 T No H1 3319 Narooma, NSW, AUS; Jan 12, 1993 T M F B B 2002, M 2003 H1 3321 Wapengo Ck., NSW, AUS; Jan 11, 1993 T No H1 3322 Merimbula, NSW, AUS; Jan 12, 1993 F No H1 3330 Garden Island, SA, AUS; Jan 22, 1993 F No H1 3546 Brunswick Heads, NSW, AUS; Oct 23, 1995 T No H3 3562 Forster, NSW, AUS; Oct 23, 1995 T M F Only on M, at H2 17°C, not 23°C 3576 Woolooware Bay, NSW, AUS; Dec 14, 1995 T No H1 3581 Williamstown, VIC, AUS; Feb 29, 1996 T M F B F 2002 H1 3612 Jawbone Reserve, Williamstown, VIC, AUS; Jun 21, 1996 T M F B B 2004 H1 3657 Sussex Inlet, NSW, AUS; Dec 14, 1996 T M F M 2004-2006, H1 2009 3658 Sussex Inlet, NSW, AUS; Dec 14, 1996 T M F M 2001-2004, H1 B 2003, 2005, 2009 3663 Bermagui, NSW, AUS; Dec 13, 1996 T M F B B M 1999-2012 H1 3671 Merimbula, NSW, AUS; Dec 14, 1996 T M F B B M 2000-2001, H1 2003-2006 3672 Sussex Inlet, NSW, AUS; Dec 14, 1996 T No H1 3677 Rhyll, Phillip I., VIC, AUS; Dec 27, 1996 T M F B B 2002 H1 3814 Corringle Beach, VIC, AUS; Jan 1, 1998 T M F No H1 3895 American R., Kangaroo I., SA, AUS; Sep 19, 1998 T M F M 2001-2012 H1 3897 Wynyard Wharf, TAS, AUS; Sep 13, 1998 T M F M 2003-2010 H1 3900 Batman Bridge, S. of George Town, TAS, AUS; Nov 17, 1998 T M F No H1 3910 Mimosa Rock National Park, NSW, AUS; Dec 16, 1998 M 2000-2010 No data 3912 Wapengo Lake, NSW, AUS; Dec 16, 1998 T No No data 3915 Kiama, Minnamura R., NSW, AUS; Dec 19, 1998 T No No data 3916 Kiama, Minnamura R., NSW, AUS; Dec 19, 1998 T No No data 3917 Sussex Inlet, NSW, AUS; Dec 19, 1998 T F No No data 3918 Sussex Inlet, NSW, AUS; Dec 19, 1998 T No No data 3929 Tumby Bay, SA, AUS; Feb 10, 1999 T B B 2001-2006 No data 3930 Tumby Bay, SA, AUS; Feb 10, 1999 M B M B 2000-2012 No data 3931 Tumby Bay, SA, AUS; Feb 10, 1999 T M F M 2003, 2006 No data 3932 Arno Bay, SA, AUS; Feb 10, 1999 T M F B B F 2002-2012 No data 3938 Blanche Harbour, SA, AUS; Feb 10, 1999 T M F No No data 3940 Blanche Harbour, SA, AUS; Feb 10, 1999 T M F B M 2001, B 2002- No data 2012 4042 Price, SA, AUS; Jan 13, 2000 F No No data 4045 Port Clinton, SA, AUS; Jan 10, 2000 T B B 2004-2012 No data 4046 Pt. Arthur, SA, AUS; Jan 10, 2000 T No No data 4203 Woolooware Bay, NSW, AUS; Oct 15, 2001 M No No data 4600 Lehn Mesi R., Pohnpei, FSM; Feb 4, 2006 NR No H3 4629 Fefan I. Chuuk, FSM; Feb 10, 2006 NR No H3 4636 Old Taliafak Bridge, GUM; Feb 12, 2006 NR No H3 B. tenella 2751 Initao, Misamis Oriental, PHI; Oct 25, 1986 T M F F 2003-2012 No data VIC, Victoria; AUS, Australia; T, tetrasporophyte; M, male; F, female; ZFA, South Africa; NSW, New South Wales; B, bisexual; NZL, New Zealand; SA, South Australia; IDN, Indonesia; MEX, Mexico; NA, not available; WA, Western Australia; MYS, Malaysia; FSM, Micronesia; TAS, Tasmania; NR, no reproduction; GUM, Guam; PHI, Philippines.

87 http://e-algae.kr Algae 2013, 28(1): 83-92

West 2006). Galls were found on 15 of 87 (17%) isolates of West 2003, 2006, 2011) and galls were found on isolates lineages H1 and H2, exclusively with Australian isolates. from three lineages (1, 5, and 6). Again the preponder- Galls were usually associated with viable spermatangial ance of galls was found on isolates from Australia. stichidia, either on unisexual or bisexual plants, although In 26 years of culture isolate 2748 produced galls only some were evident on the non-reproductive sectors. Tet- once (May 1999) on the male spermatangial stichidia and rasporophytes and female gametophytes occasionally these were very similar in overall structure to those seen had galls. This has been true over 26 years of culture for in B. simpliciuscula. On the female of isolate 2749 galls some isolates. Galls never developed on young gameto- were noted only in 1989. By contrast, isolate 2747 from phytes, only on reproductively mature gametophytes. On the same locality never had galls. Isolate 3026 female isolate 3562 no galls formed on the tetrasporophytes, fe- formed galls at irregular intervals in 1995, 2000, and 2002 males or males grown at 23°C. However, galls developed whereas the tetrasporophyte and male showed no gall de- in the vicinity of spermatangial stichidia of males but not velopment during 22 years of culture. Isolate 3492 (Texas, on females or tetrasporophytes when grown at 17°C. Iso- USA) was isolated in 1974 and did not show galls until late 3562, having numerous galls, was placed in a culture July, 2002 and these developed primarily on cladohaptera (on a shaker and in brighter light) for 3-6 weeks with the of tetrasporophytes. male of another isolate (3108) having a long history with- Isolate 3204 female (South Africa) was collected in 1991 out galls. No galls developed on isolate 3108. This is only a and had numerous galls on procarpic lateral branches partial test of Koch’s postulates (http://en.wikipedia.org/ from 1997-2012 (Fig. 2A & B). Males and tetrasporophytes wiki/Koch’s postulates). had no galls. Isolate 4124 female (Florida, USA) was col- Galls varied in appearance and size. The initial stages lected in 2000 and developed galls with mostly colorless appeared as enlarged proliferating cells. Cell divisions living cells on the tips of vegetative laterals. Regeneration appeared random and very different from the polysipho- of viable branches frequently occurred from gall tissue nous tier cell division pattern of the normal host (Fig. 1B- (Fig. 2C & D). Galls were evident for almost 8 years (2001- E). The cells in galls were often smaller than tier cells, of 2009). Males and tetrasporophytes produced galls briefly various shapes and sizes and had enlarged vacuoles. Gall in 2002. cells were less pigmented than normal host tissue cells Galls were observed on 16 males, 4 females, 4 bisexu- (Fig. 1B). They appeared to have viable nuclei that often als, and 3 tetrasporophytes of all isolates in Table 1. were larger than tier-cell nuclei (Fig. 1F). In smaller galls the cells all appeared viable but as the galls expanded Bostrychia radicosa and the cell number increased some dead cells were seen (arrow in Fig. 1F). In some larger galls new shoots arose Isolate 4086 (Sabah, Malaysia) was obtained in Au- within the cell mass (no photo). gust 2000 and showed gall formation on the nodes and In many isolates galls were present intermittently, how- gametangial sectors of the bisexual gametophytes first in ever, galls were continuously present on the male and bi- May, 2002. Isolate 4178 (New Caledonia) males developed sexual phases of 3663 (Fig. 1F) from 1999-2012 and on the galls in August, 2012 (Fig. 3A & B). Initially the gall cells male phase of 3895 from 2001-2012 (Fig. 1B). Isolate 3932 enlarged and retained fully pigmented chloroplasts (Fig. was unusual because galls appeared on the bisexual or fe- 3A) but as the galls matured pale hypertrophied cells with male thalli but not on males from 2002-2012. enlarged vacuoles were evident (Fig. 3B). Other isolates of Electron microscopic (TEM) observations. While TEM B. radicosa from Thailand (4207) and Micronesia (4614, fixation of cellular structure was difficult our micrographs 4621, 4627, and 3662) did not develop galls. did show VLPs in gall cells of isolate 3895 (Fig. 1G & H). These VLPs are of two distinct morphologies (staining dif- Bostrychia kelanensis ferently) approximately 70-75 nm in size and hexagonal in shape. In 31 isolates of B. kelanensis from Australia, Guam, In- dia, Indonesia, Malaysia, and Micronesia only one male Bostrychia moritziana / radicans (3810) from Western Australia developed galls. Grouped cells divided and enlarged, projecting from tier cells (Fig. From 390 isolates of the B. moritziana / radicans spe- 3C) in spermatangial sectors of lateral branches. Eventu- cies-complex 24 (6%) produced galls. This species com- ally irregularly shaped masses developed numerous short plex consists of seven different lineages (Zuccarello and branch apices (Fig. 3D).

http://dx.doi.org/10.4490/algae.2013.28.1.083 88 West et al. Galls of Bostrychia

A B

C D

Fig. 2. Galls on Bostrychia moritziana / radicans isolates 3204 and 4124. (A) Habit image of 3204 female with numerous white galls on branches bearing procarps. (B) Galls at tips of procarp bearing branches. Trichogynes (tr) visible projecting from gall at lower right. (C) 4124 female with gall at tip of vegetative lateral. Two regenerating shoots developed from gall cells. (D) 4124 female gall with mass of colorless cells, a few appear dead with collapsed protoplasts but most appear to be viable living cells. Healthy, branched shoot regenerated from gall tissue. Scale bars represent: A, 1 mm; B & D, 80 µm; C, 60 µm.

89 http://e-algae.kr Algae 2013, 28(1): 83-92

A C D

E F

Fig. 3. Galls on Bostrychia radicosa, isolate 4178 (A & B); B. kelanensis, isolate 3810 (C & D); B. tenella isolate 2751 (E-G). (A) B. radicosa, elongate spermatangia bearing lateral branches with two galls visible on left. Released spermatia near branch tips also visible, small developing gall with normally pigmented cells visible on far left. (B) B. radicosa, high magnification, enlarged cells lightly pigmented with enlarged vacuoles. (C) B. kelanensis, developing gall with normally pigmented cells projecting from branch. (D) B. kelanensis, older galls with extensive branch formation. (E) B. tenella, normal procarpic branches with abundant trichogynes (tr). (F) B. tenella, numerous galls on procarpic branches, trichogynes (tr). (G) B. tenella, high magnification of gall cells, tier and cortical cells with fully pigmented chloroplasts, trichogynes (tr). Scale bars represent: A & B, 50 µm; C & D, 77 µm; E & F, 100 µm; G, 25 µm.

http://dx.doi.org/10.4490/algae.2013.28.1.083 90 West et al. Galls of Bostrychia

Bostrychia tenella also indicate that much of the transmission is vertical within a single host. We did not test gall transmission to In 86 isolates of B. tenella from many different geo- progeny of gall bearing sexual parents. graphic regions only isolate 2751 (Philippines) developed Although not tested extensively three antibiotics (Peni- galls and that was only after 17 years in culture. Galls oc- cillin G, Ciprofloxacin, and Rifampin) were routinely add- curred continuously from 2003-2012 on the procarp bear- ed to various cultures without any effect on gall presence. ing female, but never on the males or tetrasporophytes. During antibiotic treatments various bacteria were also In normal females the lateral branches were uniform in clearly present on the hosts as well. shape and heavily corticated, bearing numerous pro- The greater susceptibility of males and bisexuals to gall carps (Fig. 3E). Many gall structures appeared as irregu- formation in B. simpliciuscula may be due to the numer- lar club-shaped enlargements around the procarps (Fig. ous spermatangia formed on male branches and the fre- 3F). The variably-shaped cells contained fully pigmented quent release of spermatia opening more surface areas to chloroplasts (Fig. 3G). No branch shoot proliferation oc- attachment by viruses and bacteria. curred from these galls. We know very little about causative agents of the galls seen in Bostrychia, the effects of environmental stressors on gall formation, the transmission of the causative agent DISCUSSION or the effects of galls on the fitness of their hosts. The study of potential pathogens of marine red algae should While the presence of galls is infrequent in Bostrychia be pursued more critically. culture isolates, their similar morphology and the presence of VLP in gall cells of B. simpliciuscula implicate viruses are the causative agent. Galls have been observed ACKNOWLEDGEMENTS previously in other red algal cells with VLP suggesting them as a causative agent (Apt and Gibor 1991). Tumor- The Australian Research Council grants (A19917056 ous growth is commonly caused by viruses in higher [1999-2001]; SG0935526 [1994]; S198122824 [1998]; plants (Francki et al. 1985, Scheffer 1997). S005005 [2000]), Australian Biological Resources Study Galls were not observed in laboratory culture on any program (2002-2005), and Hermon Slade Foundation other Bostrychia species although this could be due to (2005-2007) partially supported this work. Many thanks limited sampling. We have many more isolates of the B. to Ulf Karsten and Doug McBride for help with collecting moritziana / radicans (390), B. simpliciuscula (87), and B. samples in various localities around Australia and to Alan tenella (89) than of other species but it could be that these Critchley (South Africa), Rosario Braga (Brazil), and E. K. species were more susceptible to the causative agent of Ganesan (Venezuela). gall formation. It is noteworthy that almost all species with galls lack cortication except for well-corticated B. tenella in which REFERENCES only one female (2751) had galls. We have never observed galls on any Bostrychia spe- Apt, K. & Gibor, A. 1989. Development and induction of cies in the field although we have examined thousands bacteria-associated galls on Prionitis lanceolata (Rho- of specimens, however Kützing (1865) observed and illus- dophyta). Dis. Aquat. Org. 6:151-156. trated possible galls on field specimens. Apt, K. E. 1988. Galls and tumor-like growths on marine mac- The ability of galls to form from healthy tissue sepa- roalgae. Dis. Aquat. Org. 4:211-217. rated from other gall tissue, suggests that the causative Apt, K. E. & Gibor, A. 1991. The ultrastructure of galls on the agent (possibly a virus) may be latent in cells of some red alga Gracilaria epihippisora. J. Phycol. 27:409-413. Bostrychia isolates. Latent bacteria are known in higher Ashen, J. B., Cohen, J. D. & Goff, L. J. 1999. GC-SIM-MS de- plants (Francki et al. 1985), and the stimulation of their tection and quantification of free indole-3-acetic acid effects (e.g., cell proliferation) could be due to stressors in bacterial galls on the marine alga Prionitis lanceolata in the cells. This is seen in our experiments in which galls (Rhodophyta). J. Phycol. 35:493-500. were induced in low temperature conditions. The inabil- Ashen, J. B. & Goff, L. J. 1996. Molecular identification of a ity of galls to be transmitted from one isolate to another bacterium associated with gall formation in the marine in Bostrychia and in Gracilaria (Apt and Gibor 1991) may red alga Prionitis lanceolata. J. Phycol. 32:286-297.

91 http://e-algae.kr Algae 2013, 28(1): 83-92

Ashen, J. B. & Goff, L. J. 1998. Galls on the marine red alga Spurr, A. R. 1969. A low-viscosity epoxy resin embedding me- Prionitis lanceolata (Halymeniaceae): specific induction dium for electron microscopy. J. Ultrastruct. Res. 26:31- and subsequent development of an algal-bacterial sym- 43. biosis. Am. J. Bot. 85:1710-1721. Tsekos, I. 1982. Tumour-like growths induced by bacteria in Ashen, J. B. & Goff, L. J. 2000. Molecular and ecological evi- the thallus of a red alga, Gigartina teedii (Roth) Lamour. dence for species specificity and coevolution in a group Ann. Bot. 49:123-126. of marine algal-bacterial symbioses. Appl. Environ. Mi- West, J. A. 2005. Long term macroalgal culture maintenance. crobiol. 66:3024-3030. In Andersen, R. A. (Ed.) Algal Culturing Techniques. Aca- Correa, J. A., Flores, V. & Sánchez, P. 1993. Deformative dis- demic Press, New York, pp. 157-163. ease in Iridaea laminarioides (Rhodophyta): gall develo- West, J. A. & Zuccarello, G. C. 1999. Biogeography of sexual ment associated with an endophytic cyanobacterium. J. and asexual populations in Bostrychia moritziana (Rho- Phycol. 29:853-860. domelaceae, Rhodophyta). Phycol. Res. 47:115-123. Francki, R. I. B., Milne, R. G. & Hatta, T. 1985. Atlas of plant Zuccarello, G. C. 2008. A fungal gall of Catenella nipae (Caul- viruses. CRC Press, Boca Raton, FL, Vol. 1. 222 pp, Vol. acanthaceae, Rhodophyta) and a review of Catenelloco- 2. 284 pp. lax leeuwenii. Bot. Mar. 51:436-440. King, R. J. & Puttock, C. F. 1989. Morphology and taxonomy Zuccarello, G. C. & West, J. A. 2003. Multiple cryptic spe- of Bostrychia and Stictosiphonia (Rhodomelaceae/Rho- cies: molecular diversity and reproductive isolation in dophyta). Aust. Syst. Bot. 2:1-73. the Bostrychia radicans/B. moritziana complex (Rho- Kützing, F. T. 1865. Tabulae phycologicae. Abbildungen der domelaceae, Rhodophyta) with focus on North Ameri- Tange. Vol. 15. W. Koehne, Nordhausen, 36 pp. can isolates. J. Phycol. 39:948-959. McBride, D. L., Kugrens, P. & West, J. A. 1974. Light and elec- Zuccarello, G. C. & West, J. A. 2006. Molecular phylogeny of tron microscopic observations on red algal galls. Proto- the subfamily Bostrychioideae (Ceramiales, Rhodophy- plasma 79:249-264. ta): subsuming Stictosiphonia and highlighting poly- Pueschel, C. M. 1995. Rod-shaped virus-like particles in the phyly in species of Bostrychia. Phycologia 45:24-36. endoplasmic reticulum of Audouinella saviana (Acro- Zuccarello, G. C. & West, J. A. 2011. Insights into evolution chaetiales, Rhodophyta). Can. J. Bot. 73:1974-1980. and speciation in the red alga Bostrychia: 15 years of re- Reynolds, E. S. 1963. The use of lead citrate at high pH as an search. Algae 26:21-32. electron-opaque stain in electron microscopy. J. Cell Zuccarello, G. C., West, J. A., Karsten, U. & King, R. J. 1999. Biol. 17:208-212. Molecular relationships within Bostrychia tenuissima Scheffer, R. P. 1997. The nature of disease in plants. Cam- (Rhodomelaceae, Rhodophyta). Phycol. Res. 47:81-85. bridge University Press, Cambridge, 325 pp.

http://dx.doi.org/10.4490/algae.2013.28.1.083 92